Combinatorial cancer immunotherapy

ABSTRACT

Provided herein are methods and compositions for dynamically controlling and targeting multiple immunosuppressive mechanisms in cancer. Some aspects provide cells engineered to produce multiple effector molecules, each of which modulates a different immunosuppressive mechanisms of a tumor, as well as methods of using the cells to treat cancer, such as ovarian, breast, or colon cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 16/656,452filed Oct. 17, 2019, which claims the benefit of each of U.S.Provisional Application Nos. 62/747,109 filed on Oct. 17, 2018;62/747,114 filed on Oct. 17, 2018; and 62/843,180 filed May 3, 2019,each of which is hereby incorporated in its entirety by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated herein by reference inits entirety. Said ASCII copy, created on Nov. 26, 2019, is namedSTB011_SequenceListing.txt, and is 142,504 bytes in size.

BACKGROUND

There are more than 22,000 new cases of ovarian cancer and more than14,000 deaths each year in the United States (Siegel R L, et al. (2016)CA Cancer J Clin 66(1):7-30), with an estimated annual healthcare burdenof greater than $600M (Dizon D M J (2010) Gynecol Oncol 116(3)).Conventional approaches, such as chemotherapy (e.g.,carboplatin/cisplatin and/or paclitaxel), are often unable to cureovarian cancer. Approximately 70% of patients do not achieve remissionon first-line chemotherapy, and 40-50% of patients that do have aremission will relapse within three years.

Treatment of other cancers, such as breast cancer and colon cancer, isassociated with five-year survival rates of 85% and 65%, respectively.Therapies often include a combination of invasive surgeries andchemotherapies.

SUMMARY

Provided herein, in some embodiments, is a combinatorial cell-basedimmunotherapy for the targeted treatment of cancer, such as ovariancancer, breast cancer, colon cancer, lung cancer, and pancreatic cancer.This combinatorial immunotherapy relies on engineered cell circuits thatenable multifactorial modulation within and/or near a tumor (a “tumormicroenvironment (TME)”). Despite exciting advancements in combinatorialimmunotherapy, its efficacy against cancer has been limited due in partto the following challenges. It is difficult to deliver multipletherapies simultaneously to achieve maximal efficacy without triggeringsignificant side effects. It is also difficult in clinical trials todetermine the appropriate dosing and timing of multiplesystemically-administered and/or locally-injected therapies.

The combinatorial immunotherapy provided herein, however, istumor-specific and effective yet limits systemic toxicity. Thiscombinatorial immunotherapy delivers to a tumor microenvironmentmultiple immunomodulatory effector molecules from a single deliveryvehicle. The design of the delivery vehicle is optimized to improveoverall function in cancer therapy, including, but not limited to,optimization of the promoters, linkers, signal peptides, and order ofthe multiple immunomodulatory effector molecules.

Advantageously, cell circuits of the present disclosure are engineeredin mesenchymal stem cells (MSCs), which are able to selectively home totumors (including metastases), are able to produce apro-inflammatory/immunostimulatory secretome and under certainconditions an anti-inflammatory secretome, and are hypoimmunogenic.These characteristics, among others, enable their use for allogenic celltherapies, for example, without significant safety issues, side effects,or rejection.

It has been increasingly recognized that tumors are a complex interplaybetween the tumor cells and the surrounding stroma, which includes theextracellular matrix, cancer-associated stromal cells (MSCs andfibroblasts), tumor vasculature, and the immune system. The TMEsuppresses anti-tumor immune responses through multiple mechanisms thattarget both the innate and adaptive immune system of the patient. Forexample, tumors can recruit and induce regulatory T cells that suppressthe anti-tumor activity of conventional T cells by elaborating specificchemokines such as CCL22. Tumors can also express molecules that inhibitthe activity of T cells and NK cells, such as immune checkpoints such asPD-L1. Thus, targeting a single pathway is likely insufficient forachieving robust efficacy against solid tumors.

Non-limiting examples of effector molecules encompassed by the presentdisclosure include cytokines, antibodies, chemokines, nucleotides,peptides, enzymes, and oncolytic viruses. For example, MSCs may beengineered to express (and typically secrete) at least one, two, threeor more of the following effector molecules: IL-12, IL-16, IFN-β, IFN-γ,IL-2, IL-15, IL-7, IL-36γ, IL-18, IL-1β, IL-21, OX40-ligand, CD40L,anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies,anti-TGFβ antibodies, anti-TNFR2, MIP1α (CCL3), MIP1β (CCLS), CCL21, CpGoligodeoxynucleotides, and anti-tumor peptides (e.g., anti-microbialpeptides having anti-tumor activity, see, e.g., Gaspar, D. et al. FrontMicrobiol. 2013; 4: 294; Chu, H. et al. PLoS One. 2015; 10(5): e0126390,and website:aps.unmc.edu/AP/main.php).

Provided for herein is an engineered cell comprising: a) a promoter; andb) an exogenous polynucleotide sequence comprising an expressioncassette described in a formula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, E1 comprises a polynucleotide sequence encoding a firsteffector molecule, L comprises a linker polynucleotide sequence, S2comprises a polynucleotide sequence encoding a second signal peptide, E2comprises a polynucleotide sequence encoding a second effector molecule,and wherein the promoter is operably linked to the expression cassette,the first signal peptide is operably linked to the first effectormolecule, and the second signal peptide is operably linked to the secondeffector molecule. In some aspects, the engineered cell is selected fromthe group consisting of: a mesenchymal stem cell (MSC), stem cell,immune cell, natural killer (NK) cell, NKT cell, innate lymphoid cell,tumor-infiltrating lymphocyte (TIL), mast cell, eosinophil, basophil,monocyte, macrophage, neutrophil, myeloid cell, dendritic cell, T cell,CD8+ T cell, CD4+ T cell, cytotoxic T lymphocyte (CTL), viral-specific Tcell, gamma-delta T cell, T regulatory cell, and B cell.

In some aspects, the cell is a mesenchymal stem cell (MSC). In someaspects, the cell is a stem cell. In some aspects, the cell is an immunecell. In some aspects, the cell is a natural killer (NK) cell. In someaspects, the cell is a NKT cell. In some aspects, the cell is an innatelymphoid cell. In some aspects, the cell is a tumor-infiltratinglymphocyte (TIL). In some aspects, the cell is a mast cell. In someaspects, the cell is a eosinophil. In some aspects, the cell is abasophil. In some aspects, the cell is a monocyte. In some aspects, thecell is a macrophage. In some aspects, the cell is a neutrophil. In someaspects, the cell is a myeloid cell. In some aspects, the cell is adendritic cell. In some aspects, the cell is a T cell. In some aspects,the cell is a CD8+ T cell. In some aspects, the cell is a CD4+ T cell.In some aspects, the cell is a cytotoxic T lymphocyte (CTL). In someaspects, the cell is a viral-specific T cell. In some aspects, the cellis a gamma-delta T cell. In some aspects, the cell is a T regulatorycell. In some aspects, the cell is a B cell.

In some aspects, the promoter comprises an exogenous promoterpolynucleotide sequence. In some aspects, the promoter comprises anendogenous promoter. In some aspects, the promoter is operably linked tothe expression cassette such that the polynucleotides are capable ofbeing transcribed as a single polynucleotide comprising the formulaS1-E1-L-S2-E2. In some aspects, the linker polynucleotide sequence isoperably associated with the translation of the first effector moleculeand the second effector molecule as separate polypeptides. In someaspects, the linker polynucleotide sequence encodes a 2A ribosomeskipping tag. In some aspects, the 2A ribosome skipping tag is selectedfrom the group consisting of: P2A, T2A, E2A, and F2A. In some aspects,the linker polynucleotide sequence encodes a T2A ribosome skipping tag.In some aspects, the linker polynucleotide sequence encodes an InternalRibosome Entry Site (IRES). In some aspects, the linker polynucleotidesequence encodes a cleavable polypeptide. In some aspects, the cleavablepolypeptide comprises a Furin recognition polypeptide sequence. In someaspects, the linker polynucleotide sequence further encodes aGly-comprising. Ser-comprising, or Gly-Ser comprising polypeptidesequence, e.g., a Gly-Ser-Gly polypeptide sequence. In some aspects, thelinker polynucleotide sequence encodes a Furin recognition polypeptidesequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosomeskipping tag in a Furin:Gly-Ser-Gly:T2A orientation from N-terminus toC-terminus.

In some aspects, the linker polynucleotide sequence encodes a secondpromoter, wherein the promoter is operably linked to the expressioncassette such that a first polynucleotide comprising the formula S1-E1is capable of being transcribed, wherein the second promoter is operablylinked to the expression cassette such that a second polynucleotidecomprising the formula S2-E2 is capable of being transcribed, andwherein the first and the second polynucleotide are separatepolynucleotides. In some aspects, the promoter and the second promoterare identical. In some aspects, the promoter and the second promoter aredifferent.

In some aspects, the engineered cell is HLA-typed with reference to asubject in need of therapeutic treatment. In some aspects, theengineered cell is a human cell. In some aspects, the human cell is anisolated cell from a subject, e.g., the subject who will receive thecell. In some aspects, the isolated cell is isolated from a tissueconsisting of the group of: bone marrow, adipose tissue, the umbilicalcord, fetal liver, muscle, and lung tissue. In some aspects, theengineered cell is a cultured cell.

In some aspects, the engineered MSC comprises a cellular markerphenotype comprising the cellular markers CD105+, CD73+, and CD90+. Insome aspects, the cellular marker phenotype further comprises aphenotype lacking or substantially lacking one or more cellular markersselected from the group consisting of: CD45, CD34, CD14, CD11b, CD79a,CD19, HLA class II, and combinations thereof. In some aspects, theengineered MSC comprises a cellular marker phenotype comprising CD105+,CD73+, CD90+, CD45−, CD34−, CD14−; a cellular marker phenotypecomprising CD105+, CD73+, CD90+, CD11b−, CD79α−; a cellular markerphenotype comprising CD105+, CD73+, CD90+, CD19-, HLA class II−; or acellular marker phenotype comprising CD73+, CD90+, CD105+, and CD166+,CD11b−, CD14−, CD19−, CD34−, CD45−, and HLA−DR−. In some aspects, thecellular marker phenotype is determined or has been determined byflow-cytometry.

In some aspects, the engineered cell comprises a T cell. In someaspects, the engineered cell comprises a NK cell. In some aspects, theengineered cell comprises a NKT cell.

In some aspects, the cellular marker phenotype further comprises acellular marker comprising a cognate receptor or a cognate receptorligand for the first effector molecule, the second effector molecule, orthe first and second effector molecules expressed in the engineeredcells. In some aspects, the receptor is selected from the groupconsisting of: IL12RB1, IL12RB2, CCL7, and combinations thereof.

In some aspects, the promoter and/or the second promoter comprises aconstitutive promoter. In some aspects, the constitutive promoter isselected from the group consisting of: CMV, EFS, SFFV, SV40, MND, PGK,UbC, hEFlaV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94,hHSP70, hKINb, and hUBIb. In some aspects, the promoter comprises anSFFV promoter. In some aspects, the promoter and/or the second promotercomprises an inducible promoter. In some aspects, the inducible promoteris selected from the group consisting of: minP, NFkB response element,CREB response element, NFAT response element, SRF response element 1,SRF response element 2, AP1 response element, TCF-LEF response elementpromoter fusion, Hypoxia responsive element, SMAD binding element, STAT3binding site, inducer molecule responsive promoters, and tandem repeatsthereof.

In some aspects, the first signal peptide or the second signal peptidecomprises a native signal peptide native to the first effector moleculeor the second effector molecule, respectively. In some aspects, thefirst signal peptide or the second signal peptide comprises a non-nativesignal peptide non-native to the first effector molecule or the secondeffector molecule, respectively. In some aspects, the non-native signalpeptide is selected from the group consisting of: IL12, IL2, optimizedIL2, trypsiongen-2, Gaussia luciferase, CD5, human IgKVII, murineIgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidinpreprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB,osteoprotegerin, serpin E1, GROalpha, CXCL12, and IL21.

In some aspects, the first signal peptide and the second signal peptideare identical. In some aspects, the polynucleotide sequence encoding thefirst signal peptide comprises a codon optimized polynucleotidesequence. In some aspects, the first secretion polypeptide is a humanIL12 signal peptide.

In some aspects, the polynucleotide sequence encoding the second signalpeptide comprises a codon optimized polynucleotide sequence. In someaspects, the second secretion polypeptide is a human IL21 signalpeptide.

In some aspects, the first effector molecule is selected from atherapeutic class, wherein the therapeutic class is selected from thegroup consisting of: a cytokine, a chemokine, a growth factor, aco-activation molecule, a tumor microenvironment modifier a, a receptor,a ligand, an antibody, a polynucleotide, a peptide, and an enzyme.

In some aspects, the second effector molecule is selected from atherapeutic class, wherein the therapeutic class is selected from thegroup consisting of: a cytokine, a chemokine, a growth factor, aco-activation molecule, a tumor microenvironment modifier, a receptor, aligand, an antibody, a polynucleotide, a peptide, and an enzyme. In someaspects, the therapeutic class of the first effector molecule and thesecond effector molecule are different.

In some aspects, the first effector molecule and/or the second effectormolecule is a modified effector molecule. In some aspects, the firsteffector molecule and/or the second effector molecule is modified tocomprises a cell membrane tethering domain. In some aspects, the cellmembrane tethering domain comprises a transmembrane-intracellular domainor a transmembrane domain. In some aspects, the cell membrane tetheringdomain comprises a cell surface receptor, or a cell membrane-boundportion thereof. In some aspects, the modified effector molecule is afusion protein that comprises the cell surface receptor, or a cellmembrane-bound portion thereof. In some aspects, the modified effectormolecule further comprises a linker between the effector molecule andthe cell membrane tethering domain. In some aspects, when expressed themodified effector molecule is tethered to a cell membrane of theengineered cell.

In some aspects, the cytokine is selected from the group consisting of:IL12, IL7, IL21, IL18, IL15, Type I interferons, and Interferon-gamma.In some aspects, the IL12 cytokine is an IL12p70 fusion protein. In someaspects, the chemokine is selected from the group consisting of: CCL21a,CXCL10, CXCL11, CXCL13, CXCL10-11 fusion, CCL19, CXCL9, and XCL1. Insome aspects, the growth factor is selected from the group consistingof: Flt3L and GM-CSF. In some aspects, the co-activation molecule isselected from the group consisting of: 4-1BBL and CD40L. In someaspects, the tumor microenvironment modifier is selected from the groupconsisting of: adenosine deaminase, TGFbeta inhibitors, immunecheckpoint inhibitors, VEGF inhibitors, and HPGE2. In some aspects, theTGFbeta inhibitors are selected from the group consisting of: ananti-TGFbeta peptide, an anti-TGFbeta antibody, a TGFb-TRAP, andcombinations thereof. In some aspects, the immune checkpoint inhibitorscomprise anti-PD-1 antibodies. In some aspects, the VEGF inhibitorscomprise anti-VEGF antibodies, anti-VEGF peptides, or combinationsthereof.

In some aspects, the first effector molecule and the second effectormolecule are human-derived effector molecules.

In some aspects, the first effector molecule comprises interleukin 12(IL12), for example, p35 and p40 as a dimer that is generally referredto in the art as IL-12p70. In some aspects, the first effector moleculecomprises an IL12p70 fusion protein. In some aspects, the IL12p70 fusionprotein is a human IL12p70 fusion protein. In some aspects, the humanIL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 137.In some aspects, the human IL12 comprises the p35 subunit indicated inSEQ ID NO: 137. In some aspects, the human IL12 comprises the p40subunit indicated in SEQ ID NO: 137.

In some aspects, the second effector molecule comprises CCL21a. In someaspects, the CCL21a is a human CCL21a. In some aspects, the secondeffector molecule comprises IL7. In some aspects, the IL7 is a humanIL7. In some aspects, the second effector molecule comprises IL21. Insome aspects, the IL21 is a human IL21.

In some aspects, the expression cassette further comprises an E3comprising a polynucleotide sequence encoding a third effector molecule.In some aspects, the third effector molecule comprises Flt3L. In someaspects, the third effector molecule comprises anti-PD1. For example,anti-PD1 can be an anti-PD1 antibody. In some aspects, the expressioncassette further comprises an E4 comprising a polynucleotide sequenceencoding a fourth effector molecule. In some aspects, the fourtheffector molecule comprises adenosine deaminase. In some aspects, thethird effector molecule comprises adenosine deaminase. In some aspects,the third effector molecule comprises CD40L. In some aspects, the thirdeffector molecule comprises a CXCL10-CXCL11 fusion protein. In someaspects, the third effector molecule comprises XCL1.

In some aspects, the second effector molecule comprises Flt3L. In someaspects, the second effector molecule comprises a CXCL10-CXCL11 fusionprotein. In some aspects, the second effector molecule comprisesanti-PD1. In some aspects, the second effector molecule comprises CD40L.

In some aspects, the first effector molecule comprises interferon-betaand the second effector molecule comprises Flt3L.

In some aspects, the polynucleotide sequence encoding the first effectormolecule comprises a codon optimized polynucleotide sequence. In someaspects, the polynucleotide sequence encoding the second effectormolecule comprises a codon optimized polynucleotide sequence.

In some aspects, the engineered cell comprises a polynucleotide sequenceencoding the promoter and the expression cassette. In some aspects, theexogenous polynucleotide sequence comprises the sequence shown in SEQ IDNO: 144.

In some aspects, the exogenous polynucleotide sequence is integratedinto the genome of the engineered cell. In some aspects, the exogenouspolynucleotide sequence comprises one or more viral vectorpolynucleotide sequences.

In some aspects, the one or more viral vector polynucleotide sequencescomprise lentiviral, retroviral, retrotransposon, or adenoviralpolynucleotide sequences.

In some aspects, the expression cassette further comprises following E2,an additional exogenous polynucleotide sequence comprising a formula,oriented from 5′ to 3′, comprising:(L-S-E)_(X)wherein S comprises a polynucleotide sequence encoding a signal peptide,E comprises a polynucleotide sequence encoding an effector molecule, Lcomprises a linker polynucleotide sequence, X=1 to 20 wherein thepromoter is operably linked to the expression cassette, and wherein foreach X the corresponding signal peptide is operably associated with theeffector molecule.

Also provided for herein is an engineered cell comprising a construct,wherein the construct comprises: a) an SFFV promoter; and b) anexogenous polynucleotide sequence comprising an expression cassettedescribed in a formula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, wherein the first signal peptide is a human IL12 signalpeptide; E1 comprises a polynucleotide sequence encoding a firsteffector molecule, wherein the first effector molecule is a humanIL12p70 fusion protein; L comprises a linker polynucleotide sequence,wherein the linker polynucleotide sequence encodes a Furin recognitionpolypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2Aribosome skipping tag in a Furin:Gly-Ser-Gly:T2A orientation fromN-terminus to C-terminus; S2 comprises a polynucleotide sequenceencoding a second signal peptide, wherein the second signal peptide is ahuman IL21 signal peptide; E2 comprises a polynucleotide sequenceencoding a second effector molecule, wherein the second effectormolecule is human IL21; and wherein the SFFV promoter is operably linkedto the expression cassette, the first signal peptide is operably linkedto the first effector molecule, and the second signal peptide isoperably linked to the second effector molecule, and wherein theengineered cell is selected from the group consisting of: a mesenchymalstem cell (MSC), stem cell, immune cell, natural killer (NK) cell, NKTcell, innate lymphoid cell, tumor-infiltrating lymphocyte (TIL), mastcell, eosinophil, basophil, monocyte, macrophage, neutrophil, myeloidcell, dendritic cell, T cell, CD8+ T cell, CD4+ T cell, cytotoxic Tlymphocyte (CTL), viral-specific T cell, gamma-delta T cell, Tregulatory cell, and B cell. In some aspects, the human IL12p70 fusionprotein comprises the sequence shown in SEQ ID NO: 137. In some aspects,the human IL12p70 fusion protein comprises the sequence shown in SEQ IDNO: 138. In some aspects, polynucleotide sequence encoding the humanIL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 136.In some aspects, the human IL21 comprises the sequence shown in SEQ IDNO: 142. In some aspects, the human IL21 comprises the sequence shown inSEQ ID NO: 143. In some aspects, polynucleotide sequence encoding thehuman IL21 comprises the sequence shown in SEQ ID NO: 141. In someaspects, the linker comprises the sequence shown in SEQ ID NO: 140. Insome aspects, the linker polynucleotide sequence comprises the sequenceshown in SEQ ID NO: 139. In some aspects, the construct comprises thepolynucleotide sequence shown in SEQ ID NO: 144.

Also provided for herein is an engineered cell comprising a construct,wherein the construct comprises: a) an SFFV promoter; and b) anexogenous polynucleotide sequence comprising an expression cassettedescribed in a formula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, wherein the first signal peptide is a human IL12 signalpeptide; E1 comprises a polynucleotide sequence encoding a firsteffector molecule, wherein the first effector molecule is a humanIL12p70 fusion protein; L comprises a linker polynucleotide sequence,wherein the linker polynucleotide sequence encodes a Furin recognitionpolypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2Aribosome skipping tag in a Furin:Gly-Ser-Gly:T2A orientation fromN-terminus to C-terminus; S2 comprises a polynucleotide sequenceencoding a second signal peptide, wherein the second signal peptide is ahuman IL21 signal peptide; E2 comprises a polynucleotide sequenceencoding a second effector molecule, wherein the second effectormolecule is human IL21; and wherein the SFFV promoter is operably linkedto the expression cassette, the first signal peptide is operably linkedto the first effector molecule, and the second signal peptide isoperably linked to the second effector molecule, and wherein theengineered cell is a mesenchymal stem cell (MSC). In some aspects, thehuman IL12p70 fusion protein comprises the sequence shown in SEQ ID NO:137. In some aspects, the human IL12p70 fusion protein comprises thesequence shown in SEQ ID NO: 138. In some aspects, polynucleotidesequence encoding the human IL12p70 fusion protein comprises thesequence shown in SEQ ID NO: 136. In some aspects, the human IL21comprises the sequence shown in SEQ ID NO: 142. In some aspects, thehuman IL21 comprises the sequence shown in SEQ ID NO: 143. In someaspects, polynucleotide sequence encoding the human IL21 comprises thesequence shown in SEQ ID NO: 141. In some aspects, the linker comprisesthe sequence shown in SEQ ID NO: 140. In some aspects, the linkerpolynucleotide sequence comprises the sequence shown in SEQ ID NO: 139.In some aspects, the construct comprises the polynucleotide sequenceshown in SEQ ID NO: 144.

Also provided for herein is an engineered cell comprising a construct,wherein the construct comprises: a) an SFFV promoter; and b) anexogenous polynucleotide sequence comprising an expression cassettedescribed in a formula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, wherein the first signal peptide is a human IL12 signalpeptide; E1 comprises a polynucleotide sequence encoding a firsteffector molecule, wherein the first effector molecule is a humanIL12p70 fusion protein; L comprises a linker polynucleotide sequence,wherein the linker polynucleotide sequence encodes a Furin recognitionpolypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2Aribosome skipping tag in a Furin:Gly-Ser-Gly:T2A orientation fromN-terminus to C-terminus; S2 comprises a polynucleotide sequenceencoding a second signal peptide, wherein the second signal peptide is ahuman IL21 signal peptide; E2 comprises a polynucleotide sequenceencoding a second effector molecule, wherein the second effectormolecule is human IL21; and wherein the SFFV promoter is operably linkedto the expression cassette, the first signal peptide is operably linkedto the first effector molecule, and the second signal peptide isoperably linked to the second effector molecule, and wherein theengineered cell is a mesenchymal stem cell (MSC), wherein the MSCcomprises a cellular marker phenotype comprising CD73+, CD90+, CD105+,and CD166+, CD11b−, CD14−, CD19−, CD34−, CD45−, and HLA−DR−. In someaspects, the human IL12p70 fusion protein comprises the sequence shownin SEQ ID NO: 137. In some aspects, the human IL12p70 fusion proteincomprises the sequence shown in SEQ ID NO: 138. In some aspects,polynucleotide sequence encoding the human IL12p70 fusion proteincomprises the sequence shown in SEQ ID NO: 136. In some aspects, thehuman IL21 comprises the sequence shown in SEQ ID NO: 142. In someaspects, the human IL21 comprises the sequence shown in SEQ ID NO: 143.In some aspects, polynucleotide sequence encoding the human IL21comprises the sequence shown in SEQ ID NO: 141. In some aspects, thelinker comprises the sequence shown in SEQ ID NO: 140. In some aspects,the linker polynucleotide sequence comprises the sequence shown in SEQID NO: 139. In some aspects, the construct comprises the polynucleotidesequence shown in SEQ ID NO: 144. In some aspects, the cellular markerphenotype is determined or has been determined by flow-cytometry.

Also provided for herein is an engineered MSC comprising a construct,wherein the construct comprises: a) an SFFV promoter; and b) anexogenous polynucleotide sequence comprising an expression cassettedescribed in a formula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, wherein the first signal peptide is a human IL12 signalpeptide; E1 comprises a polynucleotide sequence encoding a firsteffector molecule, wherein the first effector molecule is a humanIL12p70 fusion protein; L comprises a linker polynucleotide sequence,wherein the linker polynucleotide sequence encodes a Furin recognitionpolypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2Aribosome skipping tag in a Furin:Gly-Ser-Gly:T2A orientation fromN-terminus to C-terminus; S2 comprises a polynucleotide sequenceencoding a second signal peptide, wherein the second signal peptide is ahuman IL21 signal peptide; E2 comprises a polynucleotide sequenceencoding a second effector molecule, wherein the second effectormolecule is human IL21; and wherein the SFFV promoter is operably linkedto the expression cassette, the first signal peptide is operably linkedto the first effector molecule, and the second signal peptide isoperably linked to the second effector molecule, and wherein theengineered MSC comprises a cellular marker phenotype comprising CD73+,CD90+, CD105+, and CD166+, CD11b−, CD14−, CD19−, CD34−, CD45−, andHLA−DR−. In some aspects, the human IL12p70 fusion protein comprises thesequence shown in SEQ ID NO: 137. In some aspects, the human IL12p70fusion protein comprises the sequence shown in SEQ ID NO: 138. In someaspects, polynucleotide sequence encoding the human IL12p70 fusionprotein comprises the sequence shown in SEQ ID NO: 136. In some aspects,the human IL21 comprises the sequence shown in SEQ ID NO: 142. In someaspects, the human IL21 comprises the sequence shown in SEQ ID NO: 143.In some aspects, polynucleotide sequence encoding the human IL21comprises the sequence shown in SEQ ID NO: 141. In some aspects, thelinker comprises the sequence shown in SEQ ID NO: 140. In some aspects,the linker polynucleotide sequence comprises the sequence shown in SEQID NO: 139. In some aspects, the construct comprises the polynucleotidesequence shown in SEQ ID NO: 144. In some aspects, the cellular markerphenotype is determined or has been determined by flow-cytometry.

Also provided for herein is an engineered cell comprising a construct,wherein the construct comprises: a) an SFFV promoter; and b) anexogenous polynucleotide sequence comprising an expression cassettedescribed in a formula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, wherein the first signal peptide is a human IL12 signalpeptide; E1 comprises a polynucleotide sequence encoding a firsteffector molecule, wherein the first effector molecule is a humanIL12p70 fusion protein; L comprises a linker polynucleotide sequence,wherein the linker polynucleotide sequence encodes a Furin recognitionpolypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2Aribosome skipping tag in a Furin:Gly-Ser-Gly:T2A orientation fromN-terminus to C-terminus; S2 comprises a polynucleotide sequenceencoding a second signal peptide, wherein the second signal peptide is ahuman IL21 signal peptide; E2 comprises a polynucleotide sequenceencoding a second effector molecule, wherein the second effectormolecule is human IL21; and wherein the SFFV promoter is operably linkedto the expression cassette, the first signal peptide is operably linkedto the first effector molecule, and the second signal peptide isoperably linked to the second effector molecule, and wherein theengineered cell is selected from the group consisting of: a mesenchymalstem cell (MSC), stem cell, immune cell, natural killer (NK) cell, NKTcell, innate lymphoid cell, tumor-infiltrating lymphocyte (TIL), mastcell, eosinophil, basophil, monocyte, macrophage, neutrophil, myeloidcell, dendritic cell, T cell, CD8+ T cell, CD4+ T cell, cytotoxic Tlymphocyte (CTL), viral-specific T cell, gamma-delta T cell, Tregulatory cell, and B cell. In some aspects, the human IL12p70 fusionprotein comprises the sequence shown in SEQ ID NO: 137. In some aspects,the human IL12p70 fusion protein comprises the sequence shown in SEQ IDNO: 138. In some aspects, polynucleotide sequence encoding the humanIL12p70 fusion protein comprises the sequence shown in SEQ ID NO: 136.In some aspects, the human IL21 comprises the sequence shown in SEQ IDNO: 142. In some aspects, the human IL21 comprises the sequence shown inSEQ ID NO: 143. In some aspects, polynucleotide sequence encoding thehuman IL21 comprises the sequence shown in SEQ ID NO: 141. In someaspects, the linker comprises the sequence shown in SEQ ID NO: 140. Insome aspects, the linker polynucleotide sequence comprises the sequenceshown in SEQ ID NO: 139. In some aspects, the construct comprises thepolynucleotide sequence shown in SEQ ID NO: 144. In some aspects, thecell is a mesenchymal stem cell (MSC). In some aspects, the cell is anatural killer (NK) cell. In some aspects, the cell is a NKT cell. Insome aspects, the cell is an innate lymphoid cell. In some aspects, thecell is a tumor-infiltrating lymphocyte (TIL). In some aspects, the cellis a mast cell. In some aspects, the cell is a eosinophil. In someaspects, the cell is a basophil. In some aspects, the cell is amonocyte. In some aspects, the cell is a macrophage. In some aspects,the cell is a neutrophil. In some aspects, the cell is a myeloid cell.In some aspects, the cell is a dendritic cell. In some aspects, the cellis a T cell. In some aspects, the cell is a CD8+ T cell. In someaspects, the cell is a CD4+ T cell. In some aspects, the cell is acytotoxic T lymphocyte (CTL). In some aspects, the cell is aviral-specific T cell. In some aspects, the cell is a gamma-delta Tcell. In some aspects, the cell is a T regulatory cell. In some aspects,the cell is a B cell. In some aspects, the cell is a human cell.

In some aspects, the engineered cell is HLA-typed with reference to asubject in need of therapeutic treatment. In some aspects, theengineered cell is a human cell. In some aspects, the human cell is anisolated cell from a subject, e.g., the subject who will receive thecell. In some aspects, the isolated cell is isolated from a tissueconsisting of the group of: bone marrow, adipose tissue, the umbilicalcord, fetal liver, muscle, and lung tissue. In some aspects, theengineered cell is a cultured cell.

In some aspects, the engineered MSC comprises a cellular markerphenotype comprising the cellular markers CD105+, CD73+, and CD90+. Insome aspects, the cellular marker phenotype further comprises aphenotype lacking or substantially lacking one or more cellular markersselected from the group consisting of: CD45, CD34, CD14, CD11b, CD79a,CD19, HLA class II, and combinations thereof. In some aspects, theengineered MSC comprises a cellular marker phenotype comprising CD105+,CD73+, CD90+, CD45−, CD34−, CD14−; a cellular marker phenotypecomprising CD105+, CD73+, CD90+, CD11b−, CD79α−; a cellular markerphenotype comprising CD105+, CD73+, CD90+, CD19−, HLA class II−; or acellular marker phenotype comprising CD73+, CD90+, CD105+, and CD166+,CD11b−, CD14−, CD19−, CD34−, CD45−, and HLA−DR−. In some aspects, thecellular marker phenotype is determined or has been determined byflow-cytometry.

In some aspects, the engineered cell comprises a T cell. In someaspects, the T cell is a CD8+ T cell, a CD4+ T cell, a cytotoxic Tlymphocyte (CTL), a viral-specific T cell, a gamma-delta T cell, or a Tregulatory cell. In some aspects, the engineered cell comprises a NKcell. In some aspects, the engineered cell comprises a NKT cell. In someaspects, the engineered cell comprises a monocyte cell. In some aspects,the engineered cell comprises a macrophage. In some aspects, theengineered cell comprises a TIL.

In some aspects, the exogenous polynucleotide sequence is integratedinto the genome of the engineered cell. In some aspects, the exogenouspolynucleotide sequence comprises one or more viral vectorpolynucleotide sequences. In some aspects, the one or more viral vectorpolynucleotide sequences comprise lentiviral, retroviral,retrotransposon, or adenoviral polynucleotide sequences. In someaspects, the one or more viral vector polynucleotide sequences compriselentiviral polynucleotide sequences.

In some aspects, the cell secretes each effector molecule. In someaspects, the first effector molecule is secreted at a ratio that is 10fold higher relative to secretion of the second effector molecule.

In some aspects, the cell further comprises an antigen recognizingreceptor. In some aspects, the antigen recognizing receptor recognizesan antigen selected from the group consisting of: 5T4, ADAMS, ADGRE2,AFP, AXL, B7-H3, B7-H4, B7-H6, C4.4, CA6, Cadherin 3, Cadherin 6, CCR1,CCR4, CD117, CD123, CD131, CD133, CD138, CD142, CD166, CD25, CD244,CD30, CD300LF, CD33, CD352, CD37, CD38, CD44, CD56, CD66e, CD70, CD71,CD74, CD79b, CD80, CD93, CEA, CEACAM5, Claudin18.2, CLEC12A, cMet,CSPG4, CTLA, DLK1, DLL3, DR5, EGFR, EMB, ENPP3, EpCAM, EphA2, Ephrin A4,ETBR, FGFR2, FGFR3, FRalpha, FRb, FLT3, GAPT, GCC, GD2, GFRa4, gpA33,GPC3, gpNBM, GPRC5, HER2, IL-1RAP, IL-13R, IL-13Ra, IL-13Ra2, IL-8,IL-15, IL1RAP, Integrin aV, KIT, L1CAM, LAMP1, LAT2, Lewis Y, LeY,LILRA2, LILRB2, LIV-1, LRRC, LY6E, MCSP, Mesothelin, MLC1, MS4A3, MUC1,MUC16, MUC1C, MYADM, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY ESO 1, Ovarin,P-cadherin, pan-Erb2, PIEZO1, PRAM1, PSCA, PSMA, PTK7, ROR1, S Aures,SCT, SLAMF7, SLC22A16, SLC17A9, SLITRK6, SPNS3, SSTR2, STEAP1, Survivin,TDGF1, TIM1, TROP2, VSTM1, and WT1.

In some aspects, the antigen recognizing receptor comprises anantigen-binding domain. In some aspects, the antigen-binding domaincomprises an antibody, an antigen-binding fragment of an antibody, aF(ab) fragment, a F(ab′) fragment, a single chain variable fragment(scFv), or a single-domain antibody (sdAb). In some aspects, theantigen-binding domain comprises a single chain variable fragment(scFv). In some aspects, the scFv comprises a heavy chain variabledomain (VH) and a light chain variable domain (VL). In some aspects, theVH and VL are separated by a peptide linker. In some aspects, the scFvcomprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavychain variable domain, L is the peptide linker, and VL is the lightchain variable domain.

In some aspects, the antigen recognizing receptor is a chimeric antigenreceptor (CAR) or T cell receptor (TCR). In some aspects, the antigenrecognizing receptor is a chimeric antigen receptor (CAR). In someaspects, the CAR comprises one or more intracellular signaling domains,and the one or more intracellular signaling domains are selected fromthe group consisting of: a CD3zeta-chain intracellular signaling domain,a CD97 intracellular signaling domain, a CD11a-CD18 intracellularsignaling domain, a CD2 intracellular signaling domain, an ICOSintracellular signaling domain, a CD27 intracellular signaling domain, aCD154 intracellular signaling domain, a CD8 intracellular signalingdomain, an OX40 intracellular signaling domain, a 4-1BB intracellularsignaling domain, a CD28 intracellular signaling domain, a ZAP40intracellular signaling domain, a CD30 intracellular signaling domain, aGITR intracellular signaling domain, an HVEM intracellular signalingdomain, a DAP10 intracellular signaling domain, a DAP12 intracellularsignaling domain, and a MyD88 intracellular signaling domain. In someaspects, the CAR comprises a transmembrane domain, and the transmembranedomain is selected from the group consisting of: a CD8 transmembranedomain, a CD28 transmembrane domain a CD3zeta-chain transmembranedomain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, anOX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembranedomain, a 2B4 transmembrane domain, and a BTLA transmembrane domain. Insome aspects, the CAR comprises a spacer region between theantigen-binding domain and the transmembrane domain.

Also provided for herein is a population of cells, the population ofcells comprising any of the engineered cells described herein. In someaspects, the population of cells is enriched for the engineered cells.

In some aspects, the first effector molecule, the second effectormolecule, or the first and second effector molecules expressed in theengineered cells promotes increased growth, viability, or growth andviability relative to cells in the population that do not express thefirst effector molecule, the second effector molecule, or the first andsecond effector molecules. In some aspects, the first effector moleculeis IL12 or an IL12p70 fusion protein. In some aspects, the population ofcells enriched for the engineered cells express IL12 receptor β1 orincreased levels thereof, IL12 receptor β2 or increased levels thereof,or IL12 receptor β1 and IL12 receptor β2 or increased levels thereof. Insome aspects, the second effector molecule is IL21. In some aspects, thesecond effector molecule is CCL21. In some aspects, the population ofcells enriched for the engineered cells express a CCL21 receptor orincreased levels thereof. In some aspects, the CCL21 receptor is CCR7.

Also provided for herein is a method of stimulating a cell-mediatedimmune response to a tumor cell in a subject, the method comprisingadministering to a subject having a tumor a therapeutically effectivedose of any of the engineered cells or the population of cells describedherein.

Also provided for herein is a method of stimulating (e.g., inducing) animmune response, the method comprising administering to a subject atherapeutically effective dose of any of the engineered cells or thepopulation of cells described herein.

Also provided for herein is a method of providing anti-tumor immunity ina subject, the method comprising administering to a subject in needthereof a therapeutically effective dose of any of the engineered cellsany of the engineered cells or the population of cells described herein.

Also provided for herein is a method of treating a subject havingcancer, the method comprising administering to a subject having a tumora therapeutically effective dose of any of the engineered cells or thepopulation of cells described herein.

Also provided for herein is a method of reducing tumor volume in asubject, the method comprising administering to a subject having a tumora therapeutically effective dose of any of the engineered cells or thepopulation of cells described herein.

In some aspects, the engineered cell is derived from the subject. Insome aspects, the engineered cell is allogeneic with reference to thesubject.

In some aspects, the tumor is selected from the group consisting of: anadenocarcinoma, an acute myeloid leukemia (AML), an acute lymphoblasticB-cell leukemia (BALL), an acute lymphoblastic T-cell leukemia (TALL), aB-cell prolymphocytic leukemia, a bladder tumor, a brain tumor, a breasttumor, a cervical tumor, a chronic lymphocytic leukemia, a chronicmyeloid leukemia (CML), a colorectal tumor, an esophageal tumor, aglioma, a kidney tumor, a liver tumor, a lung tumor, a lymphoma, amelanoma, a mesothelioma, a myelodysplasia, an ovarian tumor, apancreatic tumor, a plasma cell myeloma, a prostate tumor, a skin tumor,a thyroid tumor, and a uterine tumor. In some aspects, the tumor is anovarian tumor. In some aspects, the tumor is a tumor located in aperitoneal space.

Also provided for herein is an engineered cell comprising: a) apromoter; and b) an exogenous polynucleotide sequence comprising anexpression cassette described in a formula, oriented from 5′ to 3′,comprising:(L-S-E)_(X)wherein S comprises a polynucleotide sequence encoding a signal peptide,E comprises a polynucleotide sequence encoding an effector molecule, Lcomprises a linker polynucleotide sequence, X=2 to 20, wherein thepromoter is operably linked to the expression cassette, wherein for thefirst iteration of the (L-S-E) unit L is absent, and wherein for each Xthe corresponding signal peptide is operably associated with theeffector molecule, and wherein the engineered cell is selected from thegroup consisting of: a mesenchymal stem cell (MSC), stem cell, immunecell, natural killer (NK) cell, NKT cell, innate lymphoid cell,tumor-infiltrating lymphocyte (TIL), mast cell, eosinophil, basophil,monocyte, macrophage, neutrophil, myeloid cell, dendritic cell, T cell,CD8+ T cell, CD4+ T cell, cytotoxic T lymphocyte (CTL), viral-specific Tcell, gamma-delta T cell, T regulatory cell, and B cell.

Also provided for herein is a population of cells comprising one or moreengineered cells, wherein the one or more engineered cells comprise: a)a promoter; and b) an exogenous polynucleotide sequence comprising anexpression cassette described in a formula, oriented from 5′ to 3′,comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, E1 comprises a polynucleotide sequence encoding a firsteffector molecule, L comprises a linker polynucleotide sequence, S2comprises a polynucleotide sequence encoding a second signal peptide, E2comprises a polynucleotide sequence encoding a second effector molecule,and wherein the promoter is operably linked to the expression cassette,the first signal peptide is operably linked to the first effectormolecule, and the second signal peptide is operably linked to the secondeffector molecule, and wherein the engineered cell is selected from thegroup consisting of: a mesenchymal stem cell (MSC), stem cell, immunecell, natural killer (NK) cell, NKT cell, innate lymphoid cell,tumor-infiltrating lymphocyte (TIL), mast cell, eosinophil, basophil,monocyte, macrophage, neutrophil, myeloid cell, dendritic cell, T cell,CD8+ T cell, CD4+ T cell, cytotoxic T lymphocyte (CTL), viral-specific Tcell, gamma-delta T cell, T regulatory cell, and B cell.

Also provided for herein is a population of cells comprising one or moreengineered cells, wherein the one or more engineered cells comprise: a)a promoter; and b) an exogenous polynucleotide sequence comprising anexpression cassette described in a formula, oriented from 5′ to 3′,comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, E1 comprises a polynucleotide sequence encoding a firsteffector molecule, L comprises a linker polynucleotide sequence, S2comprises a polynucleotide sequence encoding a second signal peptide, E2comprises a polynucleotide sequence encoding a second effector molecule,and wherein the promoter is operably linked to the expression cassette,the first signal peptide is operably linked to the first effectormolecule, and the second signal peptide is operably linked to the secondeffector molecule, and wherein the first effector molecule, the secondeffector molecule, or the first and second effector molecules expressedin the engineered cells promotes increased growth, viability, or growthand viability relative to cells in the population that do not expressthe first effector molecule, the second effector molecule, or the firstand second effector molecules, and wherein the engineered cell isselected from the group consisting of: a mesenchymal stem cell (MSC),stem cell, immune cell, natural killer (NK) cell, NKT cell, innatelymphoid cell, tumor-infiltrating lymphocyte (TIL), mast cell,eosinophil, basophil, monocyte, macrophage, neutrophil, myeloid cell,dendritic cell, T cell, CD8+ T cell, CD4+ T cell, cytotoxic T lymphocyte(CTL), viral-specific T cell, gamma-delta T cell, T regulatory cell, andB cell.

In some aspects, the one or more engineered cells express a cognatereceptor or cognate receptor ligand for the first effector molecule, thesecond effector molecule, or the first and second effector moleculesexpressed in the engineered cells. In some aspects, the first effectormolecule is IL12 or an IL12p70 fusion protein. In some aspects, thesecond effector molecule is IL21. In some aspects, the second effectormolecule is CCL21.

Also provided for herein is a population of cells comprising one or moreengineered cells, wherein the one or more engineered cells comprise aconstruct, wherein the construct comprises: a) an SFFV promoter; and b)an exogenous polynucleotide sequence comprising an expression cassettedescribed in a formula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, wherein the first signal peptide is a human IL12 signalpeptide; E1 comprises a polynucleotide sequence encoding a firsteffector molecule, wherein the first effector molecule is a humanIL12p70 fusion protein; L comprises a linker polynucleotide sequence,wherein the linker polynucleotide sequence encodes a Furin recognitionpolypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2Aribosome skipping tag in a Furin:Gly-Ser-Gly:T2A orientation fromN-terminus to C-terminus; S2 comprises a polynucleotide sequenceencoding a second signal peptide, wherein the second signal peptide is ahuman IL21 signal peptide; E2 comprises a polynucleotide sequenceencoding a second effector molecule, wherein the second effectormolecule is human IL21; and wherein the SFFV promoter is operably linkedto the expression cassette, the first signal peptide is operably linkedto the first effector molecule, and the second signal peptide isoperably linked to the second effector molecule, and wherein theengineered cell is selected from the group consisting of: a mesenchymalstem cell (MSC), stem cell, immune cell, natural killer (NK) cell, NKTcell, innate lymphoid cell, tumor-infiltrating lymphocyte (TIL), mastcell, eosinophil, basophil, monocyte, macrophage, neutrophil, myeloidcell, dendritic cell, T cell, CD8+ T cell, CD4+ T cell, cytotoxic Tlymphocyte (CTL), viral-specific T cell, gamma-delta T cell, Tregulatory cell, and B cell.

Also provided for herein is a population of cells comprising one or moreengineered cells, wherein the one or more engineered cells comprise aconstruct, wherein the construct comprises: a) an SFFV promoter; and b)an exogenous polynucleotide sequence comprising an expression cassettedescribed in a formula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, wherein the first signal peptide is a human IL12 signalpeptide; E1 comprises a polynucleotide sequence encoding a firsteffector molecule, wherein the first effector molecule is a humanIL12p70 fusion protein; L comprises a linker polynucleotide sequence,wherein the linker polynucleotide sequence encodes a Furin recognitionpolypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2Aribosome skipping tag in a Furin:Gly-Ser-Gly:T2A orientation fromN-terminus to C-terminus; S2 comprises a polynucleotide sequenceencoding a second signal peptide, wherein the second signal peptide is ahuman IL21 signal peptide; E2 comprises a polynucleotide sequenceencoding a second effector molecule, wherein the second effectormolecule is human IL21; and wherein the SFFV promoter is operably linkedto the expression cassette, the first signal peptide is operably linkedto the first effector molecule, and the second signal peptide isoperably linked to the second effector molecule, and wherein the firsteffector molecule, the second effector molecule, or the first and secondeffector molecules expressed in the engineered cells promotes increasedgrowth, viability, or growth and viability relative to cells in thepopulation that do not express the first effector molecule, the secondeffector molecule, or the first and second effector molecules, andwherein the engineered cell is selected from the group consisting of: amesenchymal stem cell (MSC), stem cell, immune cell, natural killer (NK)cell, NKT cell, innate lymphoid cell, tumor-infiltrating lymphocyte(TIL), mast cell, eosinophil, basophil, monocyte, macrophage,neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T cell, CD4+ Tcell, cytotoxic T lymphocyte (CTL), viral-specific T cell, gamma-delta Tcell, T regulatory cell, and B cell.

In some aspects, the human IL12p70 fusion protein comprises the sequenceshown in SEQ ID NO: 137. In some aspects, the human IL12p70 fusionprotein comprises the sequence shown in SEQ ID NO: 138. In some aspects,polynucleotide sequence encoding the human IL12p70 fusion proteincomprises the sequence shown in SEQ ID NO: 136. In some aspects, thehuman IL21 comprises the sequence shown in SEQ ID NO: 142. In someaspects, the human IL21 comprises the sequence shown in SEQ ID NO: 143.In some aspects, polynucleotide sequence encoding the human IL21comprises the sequence shown in SEQ ID NO: 141. In some aspects, thelinker comprises the sequence shown in SEQ ID NO: 140. In some aspects,the linker polynucleotide sequence comprises the sequence shown in SEQID NO: 139. In some aspects, the construct comprises the polynucleotidesequence shown in SEQ ID NO: 144.

Also provided for herein is a method of producing a population of cellsenriched for one or more receptors or receptor ligands, comprisingculturing one or more cells under conditions where the one or more cellsare contacted with a first effector molecule, a second effectormolecule, or a first and a second effector molecule, wherein thecontacted cells express one or more cognate receptors or cognatereceptor ligands for the first effector molecule, the second effectormolecule, or the first and second effector molecules, and wherein thefirst effector molecule, the second effector molecule, or the first andthe second effector molecules increase growth, viability, or growth andviability of the contacted cells relative to cells cultured in theabsence of the first effector molecule, the second effector molecule, orthe first and second effector molecules.

In some aspects, the first effector molecule, the second effectormolecule, or the first and second effector molecules are heterologouslyexpressed in one or more cells, and the one or more cells are contactedwith the first effector molecule, the second effector molecule, or thefirst and second effector molecules in an autocrine manner. In someaspects, the first effector molecule, the second effector molecule, orthe first and second effector molecules are expressed in one or moreadditional cells, and the one or more cells are contacted with the firsteffector molecule, the second effector molecule, or the first and secondeffector molecules in an paracrine manner. In some aspects, the one ormore additional cells are a feeder cells. In some aspects, the one ormore cells are cultured in media.

In some aspects, the one or more cells are contacted with the firsteffector molecule, the second effector molecule, or the first and secondeffector molecules by addition of a soluble first effector molecule, asoluble second effector molecule, or a soluble first and second effectormolecules to the media. In some aspects, the soluble first effectormolecule and/or soluble second effector molecule is a recombinanteffector molecule.In some aspects, the one or more cells are cultured under adherentconditions. In some aspects, the one or more cells are adhered onto asurface. In some aspects, the adhered cells are contacted with the firsteffector molecule, the second effector molecule, or the first and secondeffector molecules by exposing the one or more cells to first effectormolecule, the second effector molecule, or the first and second effectormolecules is immobilized on the surface.

In some aspects, the first effector molecule is IL12 or an IL12p70fusion protein. In some aspects, the population of cells is enriched forIL12 receptor β1 (IL12Rβ1), enriched for IL12 receptor β2 (IL12Rβ2), orenriched for IL12Rβ1 and IL12Rβ2. In some aspects, the population ofMSCs comprises a cellular marker phenotype comprising the cellularmarkers CD105+, CD73+, CD90+, IL12Rβ1+, and IL12Rβ2+. In some aspects,the cellular marker phenotype further comprises a phenotype lacking orsubstantially lacking one or more cellular markers selected from thegroup consisting of: CD45, CD34, CD14, CD11b, CD79α, CD19, HLA class II,and combinations thereof.

In some aspects, the population of cells comprises a cell selected fromthe group consisting of: natural killer (NK) cells, NKT cells, innatelymphoid cells, mast cells, eosinophils, basophils, monocytes,macrophages, neutrophils, and dendritic cells, T cells, CD8+ T cells,CD4+ T cells, gamma-delta T cells, and T regulatory cells, and B cells.In some aspects, the population of cells comprises a T cell, a NK cell,a NKT cell, a monocyte, a macrophage, or a myeloid derived cell.

In some aspects, the second effector molecule is IL21. In some aspects,the second effector molecule is CCL21. In some aspects, the populationof cells is enriched for CCR7. In some aspects, the population of MSCscomprises a cellular marker phenotype comprising the cellular markersCD105+, CD73+, CD90+, IL12Rβ1+, IL12Rβ2+, and CCR7+. In some aspects,the cellular marker phenotype further comprises a phenotype lacking orsubstantially lacking one or more cellular markers selected from thegroup consisting of: CD45, CD34, CD14, CD11b, CD79α, CD19, HLA class II,and combinations thereof.

Also provided for herein is a population of cells enriched for one ormore receptors or receptor ligands produced by any of the methodsdescribed herein.

Also provided for herein is one or more proteins expressed by apolynucleotide sequence, wherein the polynucleotide sequence comprisinga promoter and an expression cassette described in a formula, orientedfrom 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, E1 comprises a polynucleotide sequence encoding a firsteffector molecule, L comprises a linker polynucleotide sequence, S2comprises a polynucleotide sequence encoding a second signal peptide, E2comprises a polynucleotide sequence encoding a second effector molecule,and wherein the promoter is operably linked to the expression cassette,the first signal peptide is operably linked to the first effectormolecule, and the second signal peptide is operably linked to the secondeffector molecule.

Also provided for herein is one or more proteins expressed by apolynucleotide sequence, wherein the polynucleotide sequence comprisesan expression cassette described in a formula, oriented from 5′ to 3′,comprisingS1-E1-L-S2-E2

wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, E1 comprises a polynucleotide sequence encoding a firsteffector molecule, L comprises a linker polynucleotide sequence, S2comprises a polynucleotide sequence encoding a second signal peptide, E2comprises a polynucleotide sequence encoding a second effector molecule,the first signal peptide is operably linked to the first effectormolecule, and the second signal peptide is operably linked to the secondeffector molecule.

Also provided for herein is an isolated polynucleotide sequencecomprising a promoter and an expression cassette described in a formula,oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, E1 comprises a polynucleotide sequence encoding a firsteffector molecule, L comprises a linker polynucleotide sequence, S2comprises a polynucleotide sequence encoding a second signal peptide, E2comprises a polynucleotide sequence encoding a second effector molecule,and wherein the promoter is operably linked to the expression cassette,the first signal peptide is operably linked to the first effectormolecule, and the second signal peptide is operably linked to the secondeffector molecule.

Also provided for herein is an isolated polynucleotide sequencecomprising an expression cassette described in a formula, oriented from5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, E1 comprises a polynucleotide sequence encoding a firsteffector molecule, L comprises a linker polynucleotide sequence, S2comprises a polynucleotide sequence encoding a second signal peptide, E2comprises a polynucleotide sequence encoding a second effector molecule,the first signal peptide is operably linked to the first effectormolecule, and the second signal peptide is operably linked to the secondeffector molecule.

In some aspects, the promoter comprises an exogenous promoterpolynucleotide sequence. In some aspects, the promoter comprises anendogenous promoter. In some aspects, the promoter is operably linked tothe expression cassette such that the polynucleotides are capable ofbeing transcribed as a single polynucleotide comprising the formulaS1-E1-L-S2-E2.

In some aspects, the linker polynucleotide sequence is operablyassociated with the translation of the first effector molecule and thesecond effector molecule as separate polypeptides. In some aspects, thelinker polynucleotide sequence encodes a 2A ribosome skipping tag. Insome aspects, the 2A ribosome skipping tag is selected from the groupconsisting of: P2A, T2A, E2A, and F2A. In some aspects, the linkerpolynucleotide sequence encodes a T2A ribosome skipping tag. In someaspects, the linker polynucleotide sequence encodes an Internal RibosomeEntry Site (IRES).

In some aspects, the linker polynucleotide sequence encodes a cleavablepolypeptide. In some aspects, the cleavable polypeptide comprises aFurin recognition polypeptide sequence. In some aspects, the linkerpolynucleotide sequence further encodes a Gly-comprising.Ser-comprising, or Gly-Ser comprising polypeptide sequence, e.g., aGly-Ser-Gly polypeptide sequence. In some aspects, the linkerpolynucleotide sequence encodes a Furin recognition polypeptidesequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosomeskipping tag in a Furin:Gly-Ser-Gly:T2A orientation from N-terminus toC-terminus.

In some aspects, the linker polynucleotide sequence encodes a secondpromoter, wherein the promoter is operably linked to the expressioncassette such that a first polynucleotide comprising the formula S1-E1is capable of being transcribed, wherein the second promoter is operablylinked to the expression cassette such that a second polynucleotidecomprising the formula S2-E2 is capable of being transcribed, andwherein the first and the second polynucleotide are separatepolynucleotides. In some aspects, the promoter and the second promoterare identical. In some aspects, the promoter and the second promoter aredifferent.

In some aspects, the promoter and/or the second promoter comprises aconstitutive promoter. In some aspects, the constitutive promoter isselected from the group consisting of: CMV, EFS, SFFV, SV40, MND, PGK,UbC, hEFlaV1, hCAGG, hEFlaV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94,hHSP70, hKINb, and hUBIb. In some aspects, the promoter comprises anSFFV promoter. In some aspects, the promoter and/or the second promotercomprises an inducible promoter. In some aspects, the inducible promoteris selected from the group consisting of: minP, NFkB response element,CREB response element, NFAT response element, SRF response element 1,SRF response element 2, AP1 response element, TCF-LEF response elementpromoter fusion, Hypoxia responsive element, SMAD binding element, STAT3binding site, inducer molecule responsive promoters, and tandem repeatsthereof.

In some aspects, the first signal peptide or the second signal peptidecomprises a native signal peptide native to the first effector moleculeor the second effector molecule, respectively. In some aspects, thefirst signal peptide or the second signal peptide comprises a non-nativesignal peptide non-native to the first effector molecule or the secondeffector molecule, respectively. In some aspects, the non-native signalpeptide is selected from the group consisting of: IL12, IL2, optimizedIL2, trypsiongen-2, Gaussia luciferase, CD5, human IgKVII, murineIgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidinpreprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB,osteoprotegerin, serpin E1, GROalpha, CXCL12, and IL21. In some aspects,the first signal peptide and the second signal peptide are identical. Insome aspects, the polynucleotide sequence encoding the first signalpeptide comprises a codon optimized polynucleotide sequence.

In some aspects, the first secretion polypeptide is a human IL12 signalpeptide. In some aspects, the polynucleotide sequence encoding thesecond signal peptide comprises a codon optimized polynucleotidesequence. In some aspects, the second secretion polypeptide is a humanIL21 signal peptide.

In some aspects, the first effector molecule is selected from atherapeutic class, wherein the therapeutic class is selected from thegroup consisting of: a cytokine, a chemokine, a growth factor, aco-activation molecule, a tumor microenvironment modifier a, a receptor,a ligand, an antibody, a polynucleotide, a peptide, and an enzyme. Insome aspects, the second effector molecule is selected from atherapeutic class, wherein the therapeutic class is selected from thegroup consisting of: a cytokine, a chemokine, a growth factor, aco-activation molecule, a tumor microenvironment modifier, a receptor, aligand, an antibody, a polynucleotide, a peptide, and an enzyme. In someaspects, the therapeutic class of the first effector molecule and thesecond effector molecule are different. In some aspects, the firsteffector molecule and/or the second effector molecule is a modifiedeffector molecule.

In some aspects, the first effector molecule and/or the second effectormolecule is modified to comprises a cell membrane tethering domain. Insome aspects, the cell membrane tethering domain comprises atransmembrane-intracellular domain or a transmembrane domain. In someaspects, the cell membrane tethering domain comprises a cell surfacereceptor, or a cell membrane-bound portion thereof. In some aspects, themodified effector molecule is a fusion protein that comprises the cellsurface receptor, or a cell membrane-bound portion thereof. In someaspects, the modified effector molecule further comprises a linkerbetween the effector molecule and the cell membrane tethering domain. Insome aspects, when expressed in a cell, the modified effector moleculeis tethered to a cell membrane of the cell.

In some aspects, the cytokine is selected from the group consisting of:IL12, IL7, IL21, IL18, IL15, Type I interferons, and Interferon-gamma.In some aspects, the IL12 cytokine is an IL12p70 fusion protein. In someaspects, the chemokine is selected from the group consisting of: CCL21a,CXCL10, CXCL11, CXCL13, CXCL10-11 fusion, CCL19, CXCL9, and XCL1. Insome aspects, the growth factor is selected from the group consistingof: Flt3L and GM-CSF. In some aspects, the co-activation molecule isselected from the group consisting of: 4-1BBL and CD40L. In someaspects, the tumor microenvironment modifier is selected from the groupconsisting of: adenosine deaminase, TGFbeta inhibitors, immunecheckpoint inhibitors, VEGF inhibitors, and HPGE2. In some aspects, theTGFbeta inhibitors are selected from the group consisting of: ananti-TGFbeta peptide, an anti-TGFbeta antibody, a TGFb-TRAP, andcombinations thereof. In some aspects, the immune checkpoint inhibitorscomprise anti-PD-1 antibodies. In some aspects, the VEGF inhibitorscomprise anti-VEGF antibodies, anti-VEGF peptides, or combinationsthereof.

In some aspects, the first effector molecule and the second effectormolecule are human-derived effector molecules.

In some aspects, the first effector molecule comprises IL12. In someaspects, the first effector molecule comprises an IL12p70 fusionprotein. In some aspects, the IL12p70 fusion protein is a human IL12p70fusion protein.

In some aspects, the second effector molecule comprises CCL21a. In someaspects, the CCL21a is a human CCL21a. In some aspects, the secondeffector molecule comprises IL7. In some aspects, the IL7 is a humanIL7. In some aspects, the second effector molecule comprises IL21. Insome aspects, the IL21 is a human IL21.

In some aspects, the expression cassette further comprises an E3comprising a polynucleotide sequence encoding a third effector molecule.In some aspects, the third effector molecule comprises Flt3L. In someaspects, the third effector molecule comprises anti-PD1.

In some aspects, the expression cassette further comprises an E4comprising a polynucleotide sequence encoding a fourth effectormolecule. In some aspects, the fourth effector molecule comprisesadenosine deaminase.

In some aspects, the third effector molecule comprises adenosinedeaminase. In some aspects, the third effector molecule comprises CD40L.In some aspects, the third effector molecule comprises a CXCL10-CXCL11fusion protein. In some aspects, the third effector molecule comprisesXCL1.

In some aspects, the second effector molecule comprises Flt3L. In someaspects, the second effector molecule comprises a CXCL10-CXCL11 fusionprotein. In some aspects, the second effector molecule comprisesanti-PD1. In some aspects, the second effector molecule comprises CD40L.

In some aspects, the first effector molecule comprises interferon-betaand the second effector molecule comprises Flt3L.

In some aspects, the polynucleotide sequence encoding the first effectormolecule comprises a codon optimized polynucleotide sequence. In someaspects, the polynucleotide sequence encoding the second effectormolecule comprises a codon optimized polynucleotide sequence.

In some aspects, the human IL12p70 fusion protein comprises the sequenceshown in SEQ ID NO: 137. In some aspects, the human IL12p70 fusionprotein comprises the sequence shown in SEQ ID NO: 138. In some aspects,polynucleotide sequence encoding the human IL12p70 fusion proteincomprises the sequence shown in SEQ ID NO: 136. In some aspects, thehuman IL21 comprises the sequence shown in SEQ ID NO: 142. In someaspects, the human IL21 comprises the sequence shown in SEQ ID NO: 143.In some aspects, polynucleotide sequence encoding the human IL21comprises the sequence shown in SEQ ID NO: 141. In some aspects, thelinker comprises the sequence shown in SEQ ID NO: 140. In some aspects,the linker polynucleotide sequence comprises the sequence shown in SEQID NO: 139. In some aspects, the construct comprises the polynucleotidesequence shown in SEQ ID NO: 144.

Also provided for herein is an exogenous polynucleotide sequencecomprising an SFFV promoter and an expression cassette described in aformula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, wherein the first signal peptide is a human IL12 signalpeptide; E1 comprises a polynucleotide sequence encoding a firsteffector molecule, wherein the first effector molecule is a humanIL12p70 fusion protein; L comprises a linker polynucleotide sequence,wherein the linker polynucleotide sequence encodes Furin recognitionpolypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2Aribosome skipping tag in a Furin:Gly-Ser-Gly:T2A orientation fromN-terminus to C-terminus; S2 comprises a polynucleotide sequenceencoding a second signal peptide, wherein the second signal peptide is ahuman IL21 signal peptide; E2 comprises a polynucleotide sequenceencoding a second effector molecule, wherein the second effectormolecule is human IL21; and wherein the SFFV promoter is operably linkedto the expression cassette, the first signal peptide is operably linkedto the first effector molecule, and the second signal peptide isoperably linked to the second effector molecule.

In some aspects, the human IL12p70 fusion protein comprises the sequenceshown in SEQ ID NO: 137. In some aspects, the human IL12p70 fusionprotein comprises the sequence shown in SEQ ID NO: 138. In some aspects,polynucleotide sequence encoding the human IL12p70 fusion proteincomprises the sequence shown in SEQ ID NO: 136. In some aspects, thehuman IL21 comprises the sequence shown in SEQ ID NO: 142. In someaspects, the human IL21 comprises the sequence shown in SEQ ID NO: 143.In some aspects, polynucleotide sequence encoding the human IL21comprises the sequence shown in SEQ ID NO: 141. In some aspects, thelinker comprises the sequence shown in SEQ ID NO: 140. In some aspects,the linker polynucleotide sequence comprises the sequence shown in SEQID NO: 139. In some aspects, the construct comprises the polynucleotidesequence shown in SEQ ID NO: 144.

Also provided for herein is an exogenous polynucleotide sequencecomprising an SFFV promoter and an expression cassette described in aformula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, wherein the first signal peptide is a human IL12 signalpeptide; E1 comprises a polynucleotide sequence encoding a firsteffector molecule, wherein the first effector molecule is a humanIL12p70 fusion protein; L comprises a linker polynucleotide sequence,wherein the linker polynucleotide sequence encodes Furin recognitionpolypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2Aribosome skipping tag in a Furin:Gly-Ser-Gly:T2A orientation fromN-terminus to C-terminus; S2 comprises a polynucleotide sequenceencoding a second signal peptide, wherein the second signal peptide is ahuman IL21 signal peptide; E2 comprises a polynucleotide sequenceencoding a second effector molecule, wherein the second effectormolecule is human IL21; wherein the SFFV promoter is operably linked tothe expression cassette, the first signal peptide is operably linked tothe first effector molecule, and the second signal peptide is operablylinked to the second effector molecule; wherein the promoter is operablylinked to the expression cassette such that the polynucleotides arecapable of being transcribed as a single polynucleotide comprising theformula S1-E1-L-S2-E2; and wherein the polynucleotide sequence comprisesthe polynucleotide sequence shown in SEQ ID NO: 144.

In some aspects, the exogenous polynucleotide sequence is encoded by anucleic acid selected from the group consisting of: a DNA, a cDNA, anRNA, an mRNA, and a naked plasmid.

Also provided for herein is an expression vector comprising any of theexogenous polynucleotide sequences described herein. In some aspects,the expression vector is a viral vector. In some aspects, the viralvector is a lentiviral vector.

Also provided for herein is a pharmaceutical composition comprising anyof the exogenous polynucleotide sequences described herein, and apharmaceutically acceptable carrier.

Also provided for herein is a pharmaceutical composition comprising anyof engineered cells described herein, and a pharmaceutically acceptablecarrier.

An isolated cell comprising any of the exogenous polynucleotidesequences described herein, any of the expression vectors describedherein, or any of the pharmaceutical compositions described herein.

In some aspects, the isolated cell is selected from the group consistingof: a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, acytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific Tcell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a Bcell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, amast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, amacrophage, a monocyte, a dendritic cell, an erythrocyte, a plateletcell, a human embryonic stem cell (ESC), an ESC-derived cell, apluripotent stem cell, an MSC, an induced pluripotent stem cell (iPSC),and an iPSC-derived cell.

In some aspects, the isolated cell is an MSC.

In some aspects, the exogenous polynucleotide sequence is integratedinto the genome of the cell. In some aspects, the exogenouspolynucleotide sequence comprises one or more viral vectorpolynucleotide sequences.

In some aspects, the one or more viral vector polynucleotide sequencescomprise lentiviral, retroviral, retrotransposon, or adenoviralpolynucleotide sequences. In some aspects, the one or more viral vectorpolynucleotide sequences comprise lentiviral polynucleotide sequences.

In some aspects, the engineered cell is HLA-typed with reference to asubject in need of therapeutic treatment. In some aspects, theengineered cell is a human cell. In some aspects, the human cell is anisolated cell from a subject, e.g., the subject who will receive thecell. In some aspects, the isolated cell is isolated from a tissueconsisting of the group of: bone marrow, adipose tissue, the umbilicalcord, fetal liver, muscle, and lung tissue. In some aspects, the cell isa cultured cell.

In some aspects, the MSC comprises a cellular marker phenotypecomprising the cellular markers CD105+, CD73+, and CD90+. In someaspects, the cellular marker phenotype further comprises a phenotypelacking or substantially lacking one or more cellular markers selectedfrom the group consisting of: CD45, CD34, CD14, CD11b, CD79α, CD19, HLAclass II, and combinations thereof. In some aspects, the MSC comprises acellular marker phenotype comprising CD105+, CD73+, CD90+, CD45−, CD34−,CD14−; a cellular marker phenotype comprising CD105+, CD73+, CD90+,CD11b−, CD79α−; a cellular marker phenotype comprising CD105+, CD73+,CD90+, CD19−, HLA class II−; or a cellular marker phenotype comprisingCD73+, CD90+, CD105+, and CD166+, CD11b−, CD14−, CD19−, CD34−, CD45−,and HLA−DR−. In some aspects, the cellular marker phenotype isdetermined or has been determined by flow-cytometry.

In some aspects, the cellular marker phenotype further comprises acellular marker comprising a cognate receptor or a cognate receptorligand for the first effector molecule, the second effector molecule, orthe first and second effector molecules expressed in the cell. In someaspects, the receptor is selected from the group consisting of: IL12RB1,IL12RB2, CCL7, and combinations thereof.

In some aspects, the cell secretes each effector molecule. In someaspects, the first effector molecule is secreted at a ratio that is 10fold higher relative to secretion of the second effector molecule.

In some aspects, the cell further comprises an antigen recognizingreceptor. In some aspects, the antigen recognizing receptor comprises anantigen-binding domain. In some aspects, the antigen-binding domaincomprises an antibody, an antigen-binding fragment of an antibody, aF(ab) fragment, a F(ab′) fragment, a single chain variable fragment(scFv), or a single-domain antibody (sdAb). In some aspects, theantigen-binding domain comprises a single chain variable fragment(scFv). In some aspects, the scFv comprises a heavy chain variabledomain (VH) and a light chain variable domain (VL). In some aspects, theVH and VL are separated by a peptide linker. In some aspects, the scFvcomprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavychain variable domain, L is the peptide linker, and VL is the lightchain variable domain.

In some aspects, the antigen recognizing receptor is a chimeric antigenreceptor (CAR) or T cell receptor (TCR). In some aspects, the antigenrecognizing receptor is a chimeric antigen receptor (CAR). In someaspects, the CAR comprises one or more intracellular signaling domains,and the one or more intracellular signaling domains are selected fromthe group consisting of: a CD3zeta-chain intracellular signaling domain,a CD97 intracellular signaling domain, a CD11a-CD18 intracellularsignaling domain, a CD2 intracellular signaling domain, an ICOSintracellular signaling domain, a CD27 intracellular signaling domain, aCD154 intracellular signaling domain, a CD8 intracellular signalingdomain, an OX40 intracellular signaling domain, a 4-1BB intracellularsignaling domain, a CD28 intracellular signaling domain, a ZAP40intracellular signaling domain, a CD30 intracellular signaling domain, aGITR intracellular signaling domain, an HVEM intracellular signalingdomain, a DAP10 intracellular signaling domain, a DAP12 intracellularsignaling domain, and a MyD88 intracellular signaling domain. In someaspects, the CAR comprises a transmembrane domain, and the transmembranedomain is selected from the group consisting of: a CD8 transmembranedomain, a CD28 transmembrane domain a CD3zeta-chain transmembranedomain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, anOX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembranedomain, a 2B4 transmembrane domain, and a BTLA transmembrane domain. Insome aspects, the CAR comprises a spacer region between theantigen-binding domain and the transmembrane domain.

Also provided for herein is a virus comprising any of the exogenouspolynucleotide sequences described herein or any of the expressionvectors described herein. In some aspects, the virus is selected fromthe group consisting of: a lentivirus, a retrovirus, a retrotransposon,and an adenovirus. In some aspects, the virus is a lentivirus.

Also provided for herein is a method of reducing tumor volume in asubject, the method comprising delivering to a subject having a tumor acomposition comprising cells engineered to produce multiple effectormolecules that modulate tumor-mediated immunosuppressive mechanisms, inan effective amount to reduce the volume of the tumor, wherein theengineered cells comprise: a) a promoter; and b) an exogenouspolynucleotide sequence comprising an expression cassette described in aformula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, E1 comprises a polynucleotide sequence encoding a firsteffector molecule, L comprises a linker polynucleotide sequence, S2comprises a polynucleotide sequence encoding a second signal peptide, E2comprises a polynucleotide sequence encoding a second effector molecule,and wherein the promoter is operably linked to the expression cassette,the first signal peptide is operably linked to the first effectormolecule, and the second signal peptide is operably linked to the secondeffector molecule, and wherein the engineered cell is selected from thegroup consisting of: a mesenchymal stem cell (MSC), stem cell, immunecell, natural killer (NK) cell, NKT cell, innate lymphoid cell,tumor-infiltrating lymphocyte (TIL), mast cell, eosinophil, basophil,monocyte, macrophage, neutrophil, myeloid cell, dendritic cell, T cell,CD8+ T cell, CD4+ T cell, cytotoxic T lymphocyte (CTL), viral-specific Tcell, gamma-delta T cell, T regulatory cell, and B cell.

Also provided for herein is a method of reducing tumor volume in asubject, the method comprising delivering to a subject having a tumor acomposition comprising cells engineered to produce IL12 and IL21, in aneffective amount to reduce the volume of the tumor, wherein theengineered cells comprise a construct, wherein the construct comprises:a) an SFFV promoter; and b) an exogenous polynucleotide sequencecomprising an expression cassette described in a formula, oriented from5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, wherein the first signal peptide is a human IL12 signalpeptide; E1 comprises a polynucleotide sequence encoding a firsteffector molecule, wherein the first effector molecule is a humanIL12p70 fusion protein; L comprises a linker polynucleotide sequence,wherein the linker polynucleotide sequence encodes Furin recognitionpolypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2Aribosome skipping tag in a Furin:Gly-Ser-Gly:T2A orientation fromN-terminus to C-terminus; S2 comprises a polynucleotide sequenceencoding a second signal peptide, wherein the second signal peptide is ahuman IL21 signal peptide; E2 comprises a polynucleotide sequenceencoding a second effector molecule, wherein the second effectormolecule is human IL21; and wherein the SFFV promoter is operably linkedto the expression cassette, the first signal peptide is operably linkedto the first effector molecule, and the second signal peptide isoperably linked to the second effector molecule, and wherein theengineered cell is selected from the group consisting of: a mesenchymalstem cell (MSC), stem cell, immune cell, natural killer (NK) cell, NKTcell, innate lymphoid cell, tumor-infiltrating lymphocyte (TIL), mastcell, eosinophil, basophil, monocyte, macrophage, neutrophil, myeloidcell, dendritic cell, T cell, CD8+ T cell, CD4+ T cell, cytotoxic Tlymphocyte (CTL), viral-specific T cell, gamma-delta T cell, Tregulatory cell, and B cell.

Also provided for herein is a method of stimulating (e.g., inducing) animmune response, the method comprising delivering to a subject acomposition comprising cells engineered to produce multiple effectormolecules that modulate tumor-mediated immunosuppressive mechanisms, inan effective amount to induce an immune response, wherein the engineeredcells comprise: a) a promoter; and b) an exogenous polynucleotidesequence comprising an expression cassette described in a formula,oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, E1 comprises a polynucleotide sequence encoding a firsteffector molecule, L comprises a linker polynucleotide sequence, S2comprises a polynucleotide sequence encoding a second signal peptide, E2comprises a polynucleotide sequence encoding a second effector molecule,and wherein the promoter is operably linked to the expression cassette,the first signal peptide is operably linked to the first effectormolecule, and the second signal peptide is operably linked to the secondeffector molecule, and wherein the engineered cell is selected from thegroup consisting of: a mesenchymal stem cell (MSC), stem cell, immunecell, natural killer (NK) cell, NKT cell, innate lymphoid cell,tumor-infiltrating lymphocyte (TIL), mast cell, eosinophil, basophil,monocyte, macrophage, neutrophil, myeloid cell, dendritic cell, T cell,CD8+ T cell, CD4+ T cell, cytotoxic T lymphocyte (CTL), viral-specific Tcell, gamma-delta T cell, T regulatory cell, and B cell.

Also provided for herein is a method of stimulating (e.g., inducing) animmune response in a subject, the method comprising delivering to asubject a composition comprising cells engineered to produce IL12 andIL21, in an effective amount to induce an immune response, wherein theengineered cells comprise a construct, wherein the construct comprises:a) an SFFV promoter; and b) an exogenous polynucleotide sequencecomprising an expression cassette described in a formula, oriented from5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, wherein the first signal peptide is a human IL12 signalpeptide; E1 comprises a polynucleotide sequence encoding a firsteffector molecule, wherein the first effector molecule is a humanIL12p70 fusion protein; L comprises a linker polynucleotide sequence,wherein the linker polynucleotide sequence encodes Furin recognitionpolypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2Aribosome skipping tag in a Furin:Gly-Ser-Gly:T2A orientation fromN-terminus to C-terminus; S2 comprises a polynucleotide sequenceencoding a second signal peptide, wherein the second signal peptide is ahuman IL21 signal peptide; E2 comprises a polynucleotide sequenceencoding a second effector molecule, wherein the second effectormolecule is human IL21; and wherein the SFFV promoter is operably linkedto the expression cassette, the first signal peptide is operably linkedto the first effector molecule, and the second signal peptide isoperably linked to the second effector molecule, and wherein theengineered cell is selected from the group consisting of: a mesenchymalstem cell (MSC), stem cell, immune cell, natural killer (NK) cell, NKTcell, innate lymphoid cell, tumor-infiltrating lymphocyte (TIL), mastcell, eosinophil, basophil, monocyte, macrophage, neutrophil, myeloidcell, dendritic cell, T cell, CD8+ T cell, CD4+ T cell, cytotoxic Tlymphocyte (CTL), viral-specific T cell, gamma-delta T cell, Tregulatory cell, and B cell.

In some aspects, the human IL12p70 fusion protein comprises the sequenceshown in SEQ ID NO: 137. In some aspects, the human IL12p70 fusionprotein comprises the sequence shown in SEQ ID NO: 138. In some aspects,polynucleotide sequence encoding the human IL12p70 fusion proteincomprises the sequence shown in SEQ ID NO: 136. In some aspects, thehuman IL21 comprises the sequence shown in SEQ ID NO: 142. In someaspects, the human IL21 comprises the sequence shown in SEQ ID NO: 143.In some aspects, polynucleotide sequence encoding the human IL21comprises the sequence shown in SEQ ID NO: 141. In some aspects, thelinker comprises the sequence shown in SEQ ID NO: 140. In some aspects,the linker polynucleotide sequence comprises the sequence shown in SEQID NO: 139. In some aspects, the construct comprises the polynucleotidesequence shown in SEQ ID NO: 144.

In some aspects, the method further comprises administering a checkpointinhibitor. In some aspects, the checkpoint inhibitor is an anti-PD-1antibody, anti-PD-1L antibody or an anti-CTLA-4 antibody. In someaspects, the method further comprises administering an anti-CD40antibody.

In some aspects, the tumor is selected from the group consisting of: anadenocarcinoma, an acute myeloid leukemia (AML), an acute lymphoblasticB-cell leukemia (BALL), an acute lymphoblastic T-cell leukemia (TALL), aB-cell prolymphocytic leukemia, a bladder tumor, a brain tumor, a breasttumor, a cervical tumor, a chronic lymphocytic leukemia, a chronicmyeloid leukemia (CML), a colorectal tumor, an esophageal tumor, aglioma, a kidney tumor, a liver tumor, a lung tumor, a lymphoma, amelanoma, a mesothelioma, a myelodysplasia, an ovarian tumor, apancreatic tumor, a plasma cell myeloma, a prostate tumor, a skin tumor,a thyroid tumor, and a uterine tumor. In some aspects, the tumor is anovarian tumor. In some aspects, the tumor is a tumor located in aperitoneal space.

In some aspects, the administering comprises systemic administration,intraperitoneal administration, or intratumoral administration.

In some aspects, the volume of the tumor is reduced by at least 25%relative to a control, optionally wherein the control is an unmodifiedcell. In some aspects, the volume of the tumor is reduced by at least50% relative to a control, optionally wherein the control is anunmodified cell. In some aspects, the volume of the tumor is reduced byat least 75% relative to a control, optionally wherein the control is anunmodified cell.

Also provided for herein is a method of reducing tumor volume in asubject, the method comprising delivering to a subject having a tumor acomposition capable of engineering an cell to produce multiple effectormolecules that modulate tumor-mediated immunosuppressive mechanisms, inan effective amount to reduce the volume of the tumor, wherein eachengineered cell comprises: a) a promoter; and b) an exogenouspolynucleotide sequence comprising an expression cassette described in aformula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, E1 comprises a polynucleotide sequence encoding a firsteffector molecule, L comprises a linker polynucleotide sequence, S2comprises a polynucleotide sequence encoding a second signal peptide, E2comprises a polynucleotide sequence encoding a second effector molecule,and wherein the promoter is operably linked to the expression cassette,the first signal peptide is operably linked to the first effectormolecule, and the second signal peptide is operably linked to the secondeffector molecule, and wherein the engineered cell is selected from thegroup consisting of: a mesenchymal stem cell (MSC), stem cell, immunecell, natural killer (NK) cell, NKT cell, innate lymphoid cell,tumor-infiltrating lymphocyte (TIL), mast cell, eosinophil, basophil,monocyte, macrophage, neutrophil, myeloid cell, dendritic cell, T cell,CD8+ T cell, CD4+ T cell, cytotoxic T lymphocyte (CTL), viral-specific Tcell, gamma-delta T cell, T regulatory cell, and B cell.

Also provided for herein is method of reducing tumor volume in asubject, the method comprising delivering to a subject having a tumor acomposition capable of engineering a cell to produce IL12 and IL21, inan effective amount to reduce the volume of the tumor, wherein theengineered cell comprises a construct, wherein the construct comprises:a) an SFFV promoter; and b) an exogenous polynucleotide sequencecomprising an expression cassette described in a formula, oriented from5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, wherein the first signal peptide is a human IL12 signalpeptide; E1 comprises a polynucleotide sequence encoding a firsteffector molecule, wherein the first effector molecule is a humanIL12p70 fusion protein; L comprises a linker polynucleotide sequence,wherein the linker polynucleotide sequence encodes Furin recognitionpolypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2Aribosome skipping tag in a Furin:Gly-Ser-Gly:T2A orientation fromN-terminus to C-terminus; S2 comprises a polynucleotide sequenceencoding a second signal peptide, wherein the second signal peptide is ahuman IL21 signal peptide; E2 comprises a polynucleotide sequenceencoding a second effector molecule, wherein the second effectormolecule is human IL21; and wherein the SFFV promoter is operably linkedto the expression cassette, the first signal peptide is operably linkedto the first effector molecule, and the second signal peptide isoperably linked to the second effector molecule, and wherein theengineered cell is selected from the group consisting of: a mesenchymalstem cell (MSC), stem cell, immune cell, natural killer (NK) cell, NKTcell, innate lymphoid cell, tumor-infiltrating lymphocyte (TIL), mastcell, eosinophil, basophil, monocyte, macrophage, neutrophil, myeloidcell, dendritic cell, T cell, CD8+ T cell, CD4+ T cell, cytotoxic Tlymphocyte (CTL), viral-specific T cell, gamma-delta T cell, Tregulatory cell, and B cell.

Also provided for herein is a method of stimulating (e.g., inducing) animmune response in a subject, the method comprising delivering to asubject a composition capable of engineering an cell to produce multipleeffector molecules that modulate tumor-mediated immunosuppressivemechanisms, in an effective amount to induce an immune response, whereinthe engineered cell comprises: a) a promoter; and b) an exogenouspolynucleotide sequence comprising an expression cassette described in aformula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, E1 comprises a polynucleotide sequence encoding a firsteffector molecule, L comprises a linker polynucleotide sequence, S2comprises a polynucleotide sequence encoding a second signal peptide, E2comprises a polynucleotide sequence encoding a second effector molecule,and wherein the promoter is operably linked to the expression cassette,the first signal peptide is operably linked to the first effectormolecule, and the second signal peptide is operably linked to the secondeffector molecule, and wherein the engineered cell is selected from thegroup consisting of: a mesenchymal stem cell (MSC), stem cell, immunecell, natural killer (NK) cell, NKT cell, innate lymphoid cell,tumor-infiltrating lymphocyte (TIL), mast cell, eosinophil, basophil,monocyte, macrophage, neutrophil, myeloid cell, dendritic cell, T cell,CD8+ T cell, CD4+ T cell, cytotoxic T lymphocyte (CTL), viral-specific Tcell, gamma-delta T cell, T regulatory cell, and B cell.

Also provided for herein is method of stimulating (e.g., inducing) animmune response in a subject, the method comprising delivering to asubject a composition capable of engineering a cell to produce IL12 andIL21, in an effective amount to induce an immune response, wherein theengineered cell comprises a construct, wherein the construct comprises:a) an SFFV promoter; and b) an exogenous polynucleotide sequencecomprising an expression cassette described in a formula, oriented from5′ to 3′, comprisingS1-E1-L-S2-E2wherein S1 comprises a polynucleotide sequence encoding a first signalpeptide, wherein the first signal peptide is a human IL12 signalpeptide; E1 comprises a polynucleotide sequence encoding a firsteffector molecule, wherein the first effector molecule is a humanIL12p70 fusion protein; L comprises a linker polynucleotide sequence,wherein the linker polynucleotide sequence encodes Furin recognitionpolypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2Aribosome skipping tag in a Furin:Gly-Ser-Gly:T2A orientation fromN-terminus to C-terminus; S2 comprises a polynucleotide sequenceencoding a second signal peptide, wherein the second signal peptide is ahuman IL21 signal peptide; E2 comprises a polynucleotide sequenceencoding a second effector molecule, wherein the second effectormolecule is human IL21; and wherein the SFFV promoter is operably linkedto the expression cassette, the first signal peptide is operably linkedto the first effector molecule, and the second signal peptide isoperably linked to the second effector molecule, and wherein theengineered cell is selected from the group consisting of: a mesenchymalstem cell (MSC), stem cell, immune cell, natural killer (NK) cell, NKTcell, innate lymphoid cell, tumor-infiltrating lymphocyte (TIL), mastcell, eosinophil, basophil, monocyte, macrophage, neutrophil, myeloidcell, dendritic cell, T cell, CD8+ T cell, CD4+ T cell, cytotoxic Tlymphocyte (CTL), viral-specific T cell, gamma-delta T cell, Tregulatory cell, and B cell.

In some aspects, the human IL12p70 fusion protein comprises the sequenceshown in SEQ ID NO: 137. In some aspects, the human IL12p70 fusionprotein comprises the sequence shown in SEQ ID NO: 138. In some aspects,polynucleotide sequence encoding the human IL12p70 fusion proteincomprises the sequence shown in SEQ ID NO: 136. In some aspects, thehuman IL21 comprises the sequence shown in SEQ ID NO: 142. In someaspects, the human IL21 comprises the sequence shown in SEQ ID NO: 143.In some aspects, polynucleotide sequence encoding the human IL21comprises the sequence shown in SEQ ID NO: 141. In some aspects, thelinker comprises the sequence shown in SEQ ID NO: 140. In some aspects,the linker polynucleotide sequence comprises the sequence shown in SEQID NO: 139. In some aspects, the construct comprises the polynucleotidesequence shown in SEQ ID NO: 144.

In some aspects, the composition comprises a delivery system selectedfrom the group consisting of: a viral system, a transposon system, and anuclease genomic editing system. In some aspects, the viral system isselected from the group consisting of: a lentivirus, a retrovirus, aretrotransposon, and an adenovirus. In some aspects, the nucleasegenomic editing system is selected from the group consisting of: azinc-finger system, a TALEN system, and a CRISPR system.

In some aspects, the tumor is selected from the group consisting of: anadenocarcinoma, an acute myeloid leukemia (AML), an acute lymphoblasticB-cell leukemia (BALL), an acute lymphoblastic T-cell leukemia (TALL), aB-cell prolymphocytic leukemia, a bladder tumor, a brain tumor, a breasttumor, a cervical tumor, a chronic lymphocytic leukemia, a chronicmyeloid leukemia (CML), a colorectal tumor, an esophageal tumor, aglioma, a kidney tumor, a liver tumor, a lung tumor, a lymphoma, amelanoma, a mesothelioma, a myelodysplasia, an ovarian tumor, apancreatic tumor, a plasma cell myeloma, a prostate tumor, a skin tumor,a thyroid tumor, and a uterine tumor. In some aspects, the administeringcomprises systemic administration, intraperitoneal administration, orintratumoral administration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows treatment using syngeneic and allogeneic MSCs expressingIL12p70/CCL21a in a CT26 model.

FIG. 2A shows rechallenge of tumor free mice with CT26 tumors previouslytreated using syngeneic and allogeneic MSCs expressing IL12p70/CCL21a ina CT26 model. FIG. 2A shows the treatment schematic.

FIG. 2B shows rechallenge of tumor free mice with CT26 tumors previouslytreated using syngeneic and allogeneic MSCs expressing IL12p70/CCL21a ina CT26 model. FIG. 2B shows tumor free mice rejecting the tumor implantin contrast to naïve control mice where the tumor became established.

FIG. 3 shows data indicating that intraperitoneally injected murineBM-derived MSCs (BM-MSCs) home to the tumor site of 4T1 breast cancercells in vivo. Fluorescently labeled BM-MSCs (therapeutic cells) wereinjected into mice bearing 4T1 breast tumor cells. The breast tumorcells express a luciferase reporter. The first top two panels on theleft show imaging of therapeutic cells (BM-MSCs) in mice bearing tumorson day 1 and on day 7 after injection as indicated. The third top panelon the left shows imaging of tumor cells in mice bearing tumors on day 7after injection. The bottom two panels on the left show imaging oftherapeutic cells in normal mice not bearing tumors on day 1 and on day7 after injection as indicated. A schematic showing the effect of tumorson homing of therapeutic cells is provided on the far right.

FIG. 4 shows data indicating that engineered MSCs expressing IL-12 andCCL21a induced significant tumor growth delay in an orthotopic mousemodel of breast cancer. The chart on the left shows the effects ofengineered MSCs on 4T1 breast tumor growth in mice (n=8). Each line inthe chart represents tumor volume in mice receiving intraperitonealinjection of either control MSC growth media or engineered MSCs on day 0and day 7. Mice received intraperitoneal injection of engineered MSCsexpressing IL-12 and engineered MSCs expressing CCL21a. Tumor volume wasdetermined by caliper measurements every other day. Data representmean±SEM. *p<0.05, **p<0.005 as compared to control media group. Theschematic on the right shows a timeline of treatment and the effect ofengineered MSCs expressed combinatorial genes IL-12 and CCL21a on tumorburden in treated mice.

FIG. 5A includes data indicating that engineered MSCs expressing IFN-β,IFN-γ, IL-12, CCL21a, or combinations thereof inhibit tumor growth in anorthotopic mouse model of breast cancer (4T1 triple negative breastcarcinoma). Each effector was expressed by a different MSC, and the MSCswere combined (at a 1:1 ratio) for combinatorial treatment. Each chartshows the effect of engineered MSCs expressing the indicatedimmunotherapies alone or in combination on the growth of 4T1 breasttumors in mice (n=6-8). Each line of FIG. 5A represents an individualmouse.

FIG. 5B includes data indicating that engineered MSCs expressing IFN-β,IFN-γ, IL-12, CCL21a, or combinations thereof inhibit tumor growth in anorthotopic mouse model of breast cancer (4T1 triple negative breastcarcinoma). Each effector was expressed by a different MSC, and the MSCswere combined (at a 1:1 ratio) for combinatorial treatment. The leftgraph of FIG. 5B shows the tumor weight for individual mice in eachtreatment on day 14, and the mean±SEM for each treatment group. Theright graph of FIG. 5B shows the tumor volume represented as mean±SEMfor mice receiving each treatment over time.

FIG. 6A includes data indicating that engineered MSCs expressing OX40L,TRAIL, IL15, cGAS, or combinations thereof do not inhibit tumor growthsignificantly in an orthotopic mouse model of breast cancer (4T1 triplenegative breast carcinoma). Each effector was expressed by a differentMSC, and the MSCs were combined (at a 1:1 ratio) for combinatorialtreatment. Each chart shows the effect of engineered MSCs expressing theindicated immunotherapies alone or in combination on the growth of 4T1breast tumors in mice (n=6-8). Each line of FIG. 6A represents anindividual mouse.

FIG. 6B includes data indicating that engineered MSCs expressing OX40L,TRAIL, IL15, cGAS, or combinations thereof do not inhibit tumor growthsignificantly in an orthotopic mouse model of breast cancer (4T1 triplenegative breast carcinoma). Each effector was expressed by a differentMSC, and the MSCs were combined (at a 1:1 ratio) for combinatorialtreatment. The left graph of FIG. 6B shows the tumor weight forindividual mice in each treatment, and the mean±SEM for each treatmentgroup. The right graph of FIG. 6B shows tumor volume represented asmean±SEM for mice receiving each treatment over time.

FIG. 7A includes data indicating that engineered MSCs expressing IL-12and CCL21a inhibit tumor growth in an orthotopic mouse model of breastcancer (4T1 triple negative breast carcinoma); however the addition ofanti-CD40 antibody does not reduce tumor growth. Each effector wasexpressed by a different MSC, and the MSCs were combined (at a 1:1ratio) for combinatorial treatment. Each chart shows the effect ofengineered MSCs expressing the indicated immunotherapies alone or incombination on the growth of 4T1 breast tumors in mice (n=6-8). Eachline of FIG. 7A represents an individual mouse.

FIG. 7B includes data indicating that engineered MSCs expressing IL-12and CCL21a inhibit tumor growth in an orthotopic mouse model of breastcancer (4T1 triple negative breast carcinoma); however the addition ofanti-CD40 antibody does not reduce tumor growth. Each effector wasexpressed by a different MSC, and the MSCs were combined (at a 1:1ratio) for combinatorial treatment. FIG. 7B shows the tumor weight forindividual mice in each treatment, and the mean±SEM for each treatmentgroup.

FIG. 8A includes data indicating that engineered MSCs expressing OX40L,TRAIL, IL15, HACvPD-1, or combinations thereof do not inhibit tumorgrowth significantly in an subcutaneous mouse model of breast cancer(4T1 triple negative breast carcinoma). Each effector was expressed by adifferent MSC, and the MSCs were combined (at a 1:1 ratio) forcombinatorial treatment. Each chart shows the effect of engineered MSCsexpressing the indicated immunotherapies alone or in combination on thegrowth of 4T1 breast tumors in mice (n=6-8). Each line of FIG. 8Arepresents an individual mouse.

FIG. 8B includes data indicating that engineered MSCs expressing OX40L,TRAIL, IL15, HACvPD-1, or combinations thereof do not inhibit tumorgrowth significantly in an subcutaneous mouse model of breast cancer(4T1 triple negative breast carcinoma). Each effector was expressed by adifferent MSC, and the MSCs were combined (at a 1:1 ratio) forcombinatorial treatment. The left graph of FIG. 8B shows the tumorweight for individual mice in each treatment, and the mean±SEM for eachtreatment group. The right graph of FIG. 8B shows body weightrepresented as mean±SEM for mice receiving each treatment over time.

FIG. 9A includes data indicating that engineered MSCs expressing IL-12and CCL21a inhibit tumor growth in an orthotopic mouse model of breastcancer (4T1 triple negative breast carcinoma); however the combinationof MSCs expressing CCL21a, IL-36 gamma and IL-7 does not reduce tumorgrowth. Some of the effector combinations tested, however, may causetoxicity. Each effector was expressed by a different MSC, and the MSCswere combined (at a 1:1 ratio) for combinatorial treatment. Each chartshows the effect of engineered MSCs expressing the indicatedimmunotherapies alone or in combination on the growth of 4T1 breasttumors in mice (n=6-8). Each line of FIG. 9A represents an individualmouse.

FIG. 9B includes data indicating that engineered MSCs expressing IL-12and CCL21a inhibit tumor growth in an orthotopic mouse model of breastcancer (4T1 triple negative breast carcinoma); however the combinationof MSCs expressing CCL21a, IL-36 gamma and IL-7 does not reduce tumorgrowth. Some of the effector combinations tested, however, may causetoxicity. Each effector was expressed by a different MSC, and the MSCswere combined (at a 1:1 ratio) for combinatorial treatment. FIG. 9Bshows the tumor weight for individual mice in each treatment, and themean±SEM for each treatment group.

FIG. 10A includes data from a GFP dose escalation study for toxicity andscreening. FIG. 10A shows that engineered MSCs expressing GFP do notelicit toxicity. Each effector was expressed by a different MSC, and theMSCs were combined (at a 1:1 ratio) for combinatorial treatment. Eachchart shows the effect of engineered MSCs expressing the indicatedimmunotherapies alone or in combination on the growth of 4T1 breasttumors in mice (n=6-8). Each line of FIG. 10A represents an individualmouse.

FIG. 10B includes data from a GFP dose escalation study for toxicity andscreening. Each effector was expressed by a different MSC, and the MSCswere combined (at a 1:1 ratio) for combinatorial treatment. FIG. 10Bshows the tumor weight for individual mice in each treatment, and themean±SEM for each treatment group.

FIG. 11A shows that engineered human MSCs do not home to mouse 4T1tumors. Each line of FIG. 11A represents an individual mouse.

FIG. 11B shows that engineered human MSCs do not home to mouse 4T1tumors. FIG. 11B shows the tumor weight for individual mice in eachtreatment, and the mean±SEM for each treatment group.

FIG. 12 includes data showing that IL-12 and CCL21a can reduce tumorexpansion. Each line of FIG. 12 represents an individual mouse.

FIG. 13A includes data indicating that engineered MSCs expressing IL-12and CCL21 are sufficient to inhibit tumor growth in an orthotopic mousemodel of breast cancer (4T1 triple negative breast carcinoma), and theaddition of a checkpoint inhibitor (anti-PD-1 antibody or anti-CTLA-4antibody) did not increase efficacy. Each effector was expressed by adifferent MSC, and the MSCs were combined (at a 1:1 ratio) forcombinatorial treatment, and the checkpoint inhibitor was injectedseparately. Each chart shows the effect of engineered MSCs expressingthe indicated immunotherapies alone or in combination on the growth of4T1 breast tumors in mice (n=6-8). Each line of FIG. 13A represents anindividual mouse.

FIG. 13B includes data indicating that engineered MSCs expressing IL-12and CCL21 are sufficient to inhibit tumor growth in an orthotopic mousemodel of breast cancer (4T1 triple negative breast carcinoma), and theaddition of a checkpoint inhibitor (anti-PD-1 antibody or anti-CTLA-4antibody) did not increase efficacy. Each effector was expressed by adifferent MSC, and the MSCs were combined (at a 1:1 ratio) forcombinatorial treatment, and the checkpoint inhibitor was injectedseparately. FIG. 13B shows the tumor weight for individual mice in eachtreatment, and the mean±SEM for each treatment group.

FIG. 14 shows data indicating that engineered MSCs expressing IL-12 andCCL21a induced significant tumor growth delay in a mouse model ofcolorectal cancer. The graph on the left shows the effects of engineeredMSCs on CT26 colorectal tumor growth in mice (n=8). Each line in thechart represents tumor volume in mice receiving intraperitonealinjection of either control MSC growth media or engineered MSCs on day 0and day 7. Mice received intraperitoneal injection of engineered MSCsexpressing IL-12 and engineered MSCs expressing CCL21a. Tumor volume wasdetermined by caliper measurements every other day. Data representmean±SEM. *p<0.05, **p<0.005 as compared to control media group. Theschematic on the right shows a timeline of treatment and the effect ofengineered MSCs expressed combinatorial genes IL-12 and CCL21a on tumorburden in treated mice.

FIG. 15 is a graph showing tumor growth kinetics in the CT26 mouse modelto determine optimal time for dosing the engineered MSC cells.

FIG. 16A includes data indicating the effects of engineered MSCsexpressing IL-12 and CCL21a combined with anti-CD40 or anti-CTLA4antibodies on average tumor growth in a syngeneic mouse model of coloncancer. Mice bearing CT26 colon tumors were treated with one of seventreatments (n=5-6 per treatment group). MSC-IL-12+MSC-CCL21a indicatestreatment with engineered cells expressing IL-12 and with engineeredcells expressing CCL21a (at a 1:1 ratio) for combinatorial treatment.Each line of FIG. 16A represents an individual mouse.

FIG. 16B includes data indicating the effects of engineered MSCsexpressing IL-12 and CCL21a combined with anti-CD40 or anti-CTLA4antibodies on average tumor growth in a syngeneic mouse model of coloncancer. Mice bearing CT26 colon tumors were treated with one of seventreatments (n=5-6 per treatment group). MSC-IL-12+MSC-CCL21a indicatestreatment with engineered cells expressing IL-12 and with engineeredcells expressing CCL21a (at a 1:1 ratio) for combinatorial treatment.The left graph of FIG. 16B shows the tumor weight for individual mice ineach treatment, and the mean±SEM for each treatment group. The rightgraph of FIG. 16B shows the tumor volume represented as mean±SEM formice receiving each treatment over time.

FIG. 17A includes data from a dose-dependent long-term survival study.FIG. 17A shows the tumor volume of the individual group. Each line ofFIG. 17A represents an individual mouse.

FIG. 17B includes data from a dose-dependent long-term survival study.FIG. 17B shows body weight represented as mean±SEM (top left), tumorvolume represented as mean±SEM (bottom left), and survival rate (right).

FIG. 18A includes data indicating that engineered MSCs expressing IL-12,CCL21a, and either IL15 or HACvPD-1 inhibit tumor growth significantlyin a mouse model of colorectal cancer. Each effector was expressed by adifferent MSC, and the MSCs were combined (at a 1:1 ratio) forcombinatorial treatment. Each chart shows the effect of engineered MSCsexpressing the indicated immunotherapies alone or in combination on thegrowth of CT26 colorectal tumors in mice (n=6-8). Each line of FIG. 18Arepresents an individual mouse.

FIG. 18B includes data indicating that engineered MSCs expressing IL-12,CCL21a, and either IL15 or HACvPD-1 inhibit tumor growth significantlyin a mouse model of colorectal cancer. Each effector was expressed by adifferent MSC, and the MSCs were combined (at a 1:1 ratio) forcombinatorial treatment. FIG. 18B shows the tumor weight for individualmice in each treatment, and the mean±SEM for each treatment group.

FIG. 18C includes data indicating that engineered MSCs expressing IL-12,CCL21a, and either IL15 or HACvPD-1 inhibit tumor growth significantlyin a mouse model of colorectal cancer. Each effector was expressed by adifferent MSC, and the MSCs were combined (at a 1:1 ratio) forcombinatorial treatment. FIG. 18C is a representative graph of theinfiltrating immune population within the tumor microenvironment forindividual mice in each treatment, and the mean±SEM for each treatmentgroup.

FIG. 18D includes data indicating that engineered MSCs expressing IL-12,CCL21a, and either IL15 or HACvPD-1 inhibit tumor growth significantlyin a mouse model of colorectal cancer. Each effector was expressed by adifferent MSC, and the MSCs were combined (at a 1:1 ratio) forcombinatorial treatment. FIG. 18D shows the percentage of regulatory Tcells (Treg) in the total CD3 population for individual mice in eachtreatment, and the mean±SEM for each treatment group. There was asignificant decrease in the numbers of Tregs in the tumormicroenvironment treated with engineered MSC-IL2 and CCL21a.

FIG. 18E includes data indicating that engineered MSCs expressing IL-12,CCL21a, and either IL15 or HACvPD-1 inhibit tumor growth significantlyin a mouse model of colorectal cancer. Each effector was expressed by adifferent MSC, and the MSCs were combined (at a 1:1 ratio) forcombinatorial treatment. FIG. 18E correlates the percentage of immuneinfiltration with tumor weight. Samples with high lymphocytes (CD3+)were found to correlate with low tumor weight, while samples with highmyeloid (CD11b+) infiltration were correlated with higher tumor burden.

FIG. 19 shows the tumor volume for individual mice in each treatment.Efficacy was determined by tumor volume from caliper measurement everyother day.

FIG. 20 shows the tumor weight for individual mice in each treatment,and the mean±SEM for each treatment group. Efficacy was determined bytumor weight.

FIG. 21A shows the kinetics of CT26-LUC (luciferase) tumor growth in theintraperitoneal space. A CT26 cell line was injected at day 0 and three(3) mice were harvested at day 7, day 10, day 14, and day 18 todetermine the kinetics of tumor growth. The first row of FIG. 21Ameasures the mice body weight (left panel) and ROI (right panel) with anIVIS imager to monitor tumor burden. The second row monitors the tumorweight (left panel) and the ROI (right panel) of the tumor of individualmice in each group. The third row correlates the tumor weight witheither whole body ROI (left panel) or tumor ROI (right panel).

FIG. 21B shows the kinetics of CT26-LUC (luciferase) tumor growth in theintraperitoneal space. A CT26 cell line was injected at day 0 and three(3) mice were harvested at day 7, day 10, day 14, and day 18 todetermine the kinetics of tumor growth. FIG. 21B shows the immuneprofile of three (3) mice in the day 18 group to better characterize thetumor microenvironment.

FIG. 22A includes data indicating that engineered MSCs expressing IL-12and CCL21a inhibit tumor growth in a subcutaneous mouse model ofcolorectal cancer; however the combination of MSCs expressing CCL21a andIL-36 gamma or IL-7 does not reduce tumor growth. Each effector wasexpressed by a different MSC, and the MSCs were combined (at a 1:1ratio) for combinatorial treatment. Each chart shows the effect ofengineered MSCs expressing the indicated immunotherapies alone or incombination on the growth of CT26 colon tumors in mice (n=6-8). Eachline of FIG. 22A represents an individual mouse.

FIG. 22B includes data indicating that engineered MSCs expressing IL-12and CCL21a inhibit tumor growth in a subcutaneous mouse model ofcolorectal cancer; however the combination of MSCs expressing CCL21a andIL-36 gamma or IL-7 does not reduce tumor growth. Each effector wasexpressed by a different MSC, and the MSCs were combined (at a 1:1ratio) for combinatorial treatment. FIG. 22B shows the tumor weight forindividual mice in each treatment group, and the mean±SEM for eachtreatment group.

FIG. 23A includes tumor immune infiltrate statistics from the experimentrepresented by FIGS. 22A-22B. Three mice were selected from PBS, NaïveMSC, and MSC-IL12+MSC-CCL21a (combo) group to run flow cytometry toimmune profile tumor microenvironment. FIG. 23A shows a significantincrease in infiltrating CD3 and CD8 cytotoxic T population in the combogroup compared to the group dosed with naïve MSC.

FIG. 23B includes tumor immune infiltrate statistics from the experimentrepresented by FIGS. 22A-22B. Three mice were selected from PBS, NaïveMSC, and MSC-IL12+MSC-CCL21a (combo) group to run flow cytometry toimmune profile tumor microenvironment. FIG. 23B shows a significantreduction in granulocytic myeloid-derived suppressor cells (gMDSCs) andmacrophage population in the combo group compared to group treated withNaïve MSC.

FIG. 24A includes data relating to immune percentage and tumor weight,relating to the experiments represented by FIGS. 22A-22B. FIG. 24A showsthat samples with more CD3+ and CD8+ T cells (top left and top centergraph) correlate strongly with a decrease in tumor weight. These figuresalso show that samples with fewer CD11b myeloid cells, includingmacrophage, dendritic cells, and MDSC, display lower tumor burden (lowercenter and lower right graph).

FIG. 24B includes data relating to immune percentage and tumor weight,relating to the experiments represented by FIGS. 22A-22B. FIG. 24B showsthat samples with fewer CD11b myeloid cells, including macrophage,dendritic cells, and MDSC, display lower tumor burden (upper row).

FIG. 25A includes data from MSC-IL-12+CCL21a therapy in intraperitonealand subcutaneous colorectal cancer mouse models. Three different lots ofa lentiviral transduced line was tested for MSC-IL12 and CCL21a(TLOO8-3/4, TL019-01/02, and TL022-01/02; each TL number represents onelot). FIG. 25A shows that all three lots of MSC-IL12+MSC-CCL21a canreduce tumor burden in both subcutaneous and intraperitoneal model(first 5 graphs are from the SC model and last 3 are from the IP model).Tumors from all mice were collected on day 11. Each line of FIG. 25Arepresents an individual mouse.

FIG. 25B includes data from MSC-IL-12+CCL21a therapy in intraperitonealand subcutaneous colorectal cancer mouse models. Three different lots ofa lentiviral transduced line was tested for MSC-IL12 and CCL21a(TLOO8-3/4, TL019-01/02, and TL022-01/02; each TL number represents onelot). FIG. 25B shows the average tumor weight from each group, and themean±SEM for each treatment group.

FIG. 26A includes data indicating that engineered combination treatmentMSC-IL-12+MSC-CCL21a, or MSC-CCL21a+MSC-IFN-β, inhibit tumor growth in asubcutaneous mouse model of colorectal cancer; however the combinationof MSCs expressing CCL21a and s41BBL does not reduce tumor growth. Eacheffector was expressed by a different MSC, and the MSCs were combined(at a 1:1 ratio) for combinatorial treatment. Each chart shows theeffect of engineered MSCs expressing the indicated immunotherapies aloneor in combination on the growth of CT26 tumors in mice (n=6-8). Eachline of FIG. 26A represents an individual mouse.

FIG. 26B includes data indicating that engineered combination treatmentMSC-IL-12+MSC-CCL21a, or MSC-CCL21a+MSC-IFN-β, inhibit tumor growth in asubcutaneous mouse model of colorectal cancer; however the combinationof MSCs expressing CCL21a and s41BBL does not reduce tumor growth. Eacheffector was expressed by a different MSC, and the MSCs were combined(at a 1:1 ratio) for combinatorial treatment. FIG. 26B shows the tumorweight for individual mice in each treatment, and the mean for eachtreatment group. MSC-IL12+MSC-CCL21a shows best efficacy compared tomice injected with naïve MSC. Treatment efficacy was also observed inthe group treated with MSC-IFNb+MSC-CCL21a.

FIG. 27A provides additional data from the experiment represented byFIGS. 26A-26B. FIG. 27A are graphs that show immune profiles of eachgroup treated with indicated engineered MSC. A consistent decrease inmacrophage population was observed after treating withMSC-IL12+MSC-CCL21a. A general trend of increased infiltration in CD3+population and decreased infiltration in CD11b+ population was alsoobserved when compared to group treated with MSC-IL12+MSC-CCL21a againstnaïve MSC.

FIG. 27B provides additional data from the experiment represented byFIGS. 26A-26B. FIG. 27B are graphs that show immune profiles of eachgroup treated with indicated engineered MSC. A general trend ofincreased infiltration in CD3+ population and decreased infiltration inCD11b+ population was also observed when compared to group treated withMSC-IL12+MSC-CCL21a against naïve MSC.

FIG. 28A also provides_additional data from the experiment representedby FIGS. 26A-26B. FIG. 28A shows the correlation of immune infiltrationwith tumor weight.

FIG. 28B also provides additional data from the experiment representedby FIGS. 26A-26B. FIG. 28B shows the correlation of immune infiltrationwith tumor weight. Samples with low macrophage and dendritic cells havelower tumor burden (top center and top right).

FIG. 29 shows graphs combining the in vivo data from the colorectalcancer models above (FIG. 22A and FIG. 26A). The combined CT26 data fromFIG. 22A and FIG. 26A capture three groups: Tumor only (PBS), treatedwith naïve MSC, and treated with MSC-IL12+MSC-CCL21a.

FIG. 30A also shows combined data from FIG. 22A and FIG. 26A. The graphsshow the average number of immune infiltration from the flow cytometryexperiment data. Statistical significance was observed in CD8+T,demonstrating the ability of MSC-IL12+MSC-CCL21a to repolarize tumormicroenvironment and allow more cytotoxic T cell infiltration.

FIG. 30B also shows combined data from FIG. 22A and FIG. 26A. The graphsshow the average number of immune infiltration from the flow cytometryexperiment data. There was a reduction in CD11b+ myeloid populationinfiltration in the groups that were treated by MSC-IL12+MSC-CCL21a. Thedata collected show that the dendritic cells and the macrophagepopulation was statistical significance.

FIG. 31 shows the vector map of pL17D.

FIG. 32 shows MSCs engineered to express different effector moleculeseither alone or in combination and their efficacy in reducing CT26 tumorburden in an IP tumor model as assessed by BLI levels.

FIG. 33 shows MSCs engineered to express different effector moleculeseither alone or in combination and their efficacy in reducing B16F10tumor burden in an IP tumor model as assessed by BLI levels.

FIG. 34 shows the lentiviral expression vector map for expression ofhuman IL12 (p70) and human CCL21a from a single lentiviral expressionvector.

FIG. 35A shows production by engineered hMSCs of hIL12, as assessed bycytokine ELISA.

FIG. 35B shows production by engineered hMSCs of hCCL21a, as assessed bycytokine ELISA.

FIG. 36A shows a schematic of a transwell assay for assessing functionalT cell modulation by hIL12 produced from MSCs.

FIG. 36B shows a transwell assay demonstrating functional T cellmodulation by hIL12 produced from MSCs as assessed by IFNγ production.

FIG. 37A shows homing to tumors by MSCs in IP tumor-bearing mice tumorsas assessed by bioluminescence imaging. FIG. 37A shows homing in a CT26tumor model (images shown).

FIG. 37B shows homing to tumors by MSCs in IP tumor-bearing mice tumorsas assessed by bioluminescence imaging. FIG. 37B shows homing in a CT26tumor model for individual mice in each treatment, and the mean±SEM foreach treatment group (quantification summary of images shown in FIG.37A).

FIG. 37C shows homing to tumors by MSCs in IP tumor-bearing mice tumorsas assessed by bioluminescence imaging. FIG. 37C shows quantitative realtime PCR for individual mice in each treatment, and the mean±SEM foreach treatment group.

FIG. 37D shows homing to tumors by MSCs in IP tumor-bearing mice tumorsas assessed by bioluminescence imaging. FIG. 37D shows fluorescencemicroscopy against firefly luciferase.

FIG. 37E shows homing to tumors by MSCs in IP tumor-bearing mice tumorsas assessed by bioluminescence imaging. FIG. 37E shows homing in aB16F10 tumor model for individual mice in each treatment, and themean±SEM for each treatment group (quantification summary of images).

FIG. 38 shows IL12p70 expressing MSCs leading to reduction in tumorburden as assessed by BLI (top panels—images; and bottom leftpanel—individual mice in each treatment and the mean±SEM for eachtreatment group) and a complete elimination of detectableintraperitoneal tumors by tumor weight for individual mice in eachtreatment, and the mean±SEM for each treatment group (bottom rightpanel) in a CT26 IP model.

FIG. 39 shows IL12p70 expressing MSCs leading to reduction in tumorburden as assessed by BLI (top panels—images; and bottom leftpanel—individual mice in each treatment and the mean±SEM for eachtreatment group) and a complete elimination of detectableintraperitoneal tumors by tumor weight for individual mice in eachtreatment, and the mean±SEM for each treatment group (bottom rightpanel) in a B16F10 IP model.

FIG. 40A shows IL12p70/CCL21a expressing MSCs leading to reduction intumor burden as assessed by BLI in a CT26 IP model. FIG. 40A shows themean tumor burden as assessed by BLI for PBS treated (circle),MSC-Flag-Myc (“Naïve MSC” square), and IL12p70/CCL21a expressing MSCs(triangle).

FIG. 40B shows IL12p70/CCL21a expressing MSCs leading to reduction intumor burden as assessed by BLI in a CT26 IP model. FIG. 40B shows thetumor burden in individual mice as assessed by BLI for PBS treated,MSC-Flag-Myc (“Naïve MSC”), and IL12p70/CCL21a expressing MSCs (left,middle, and right panels, respectively). Each line of FIG. 40Brepresents an individual mouse.

FIG. 40C shows treatment with IL12p70/CCL21a expressing MSCs led toprolonged survival (100% survival greater than 90 days), while controltreated mice all died or were euthanized by Day 20.

FIG. 41 shows treatment with IL12p70 expressing MSCs led to prolongedsurvival.

FIG. 42A shows relative growth of genetically engineered MSCs acrossdifferent MOIs (95000, 9500, 950, or uninfected) in Donor 1.

FIG. 42B shows relative growth of genetically engineered MSCs acrossdifferent MOIs (95000, 9500, 950, or uninfected) in Donor 2.

FIG. 42C shows relative growth of genetically engineered MSCs acrossdifferent MOIs (95000, 9500, 950, or uninfected) in Donor 3.

FIG. 43 shows two independent human BM-MSC cell lines from 2 differentdonors (top and bottom row, respectively) that were transduced withconstructs containing various promoters driving EGFP expression. PercentGFP (left panels) and MFI (right panels) of engineered cells at day 25post transduction is shown.

FIG. 44 shows two independent human BM-MSC cell lines from 2 differentdonors that were transduced with constructs containing various promotersdriving EGFP expression. Shown is EGFP MFI tracked over time (day 7 today 28 post-transduction) for either the two independent human BM-MSCcell lines individually (left panel) or with data from the twoindependent human BM-MSC cell lines combined (right panel).

FIG. 45 shows secretion of IL-12p70 by engineered MSCs as assessed byELISA.

FIG. 46 shows secretion of IL-21 by engineered MSCs as assessed byELISA.

FIG. 47 shows the ratio of secreted IL-12p70 to IL-21 by engineered MSCsas assessed by ELISA.

FIG. 48 shows results of a functional reporter assay for IL-12p70 usingHEK-293T cells with a STAT4-SEAP reporter to assess cytokine productionand secretion by engineered MSCs.

FIG. 49 shows a results of a functional reporter assay for IL-21 usingintracellular phospho-flow to quantify phospho-STAT1 (left panel) andphospho-STAT3 (right panel) in NK-92 human natural killer cells toassess cytokine production and secretion by engineered MSCs.

FIG. 50 shows results of a functional reporter assay for IL-12 using aIL21R-U2OS IL21R/IL2RG dimerization reporter to assess cytokineproduction and secretion by engineered MSCs.

FIG. 51A shows MSCs engineered to express different effector moleculeseither alone or in combination and their efficacy in reducing CT26 tumorburden in an IP tumor model as assessed by BLI levels.

FIG. 51B shows MSCs engineered to express different effector moleculeseither alone or in combination and their efficacy in reducing B16F10tumor burden in an IP tumor model as assessed by BLI levels.

FIG. 52A shows efficacy of treatment using IL12p70-expressing MSCs,IL21-expressing MSCs, and the combination of IL12p70 and IL21-expressingMSCs as assessed by BLI for individual mice in each treatment, and themean±SEM for each treatment group (left panel) and by tumor weight forindividual mice in each treatment, and the mean±SEM for each treatmentgroup (right panel) in a CT26 model.

FIG. 52B shows efficacy of treatment using IL12p70-expressing MSCs,IL21-expressing MSCs, and the combination of IL12p70 and IL21-expressingMSCs in a CT26 model. FIG. 52B demonstrates the BLI luciferasemeasurements of individual mice.

FIG. 53A shows efficacy of treatment using a lower dose ofIL12p70-expressing MSCs, and the combination of IL12p70 andIL21-expressing MSCs as assessed by BLI; individual BLI measurements ofmice—left panel; summary of BLI measurements for individual mice in eachtreatment, and the mean±SEM for each treatment group—right panel).

FIG. 53B shows efficacy of treatment using a lower dose ofIL12p70-expressing MSCs, and the combination of IL12p70 andIL21-expressing MSCs. FIG. 53B shows survival curves of the treatmentgroups.

FIG. 54 shows efficacy of treatment using IL12p70-expressing MSCs,IL21-expressing MSCs, and the combination of IL12p70 and IL21-expressingMSCs as assessed by BLI for individual mice in each treatment, and themean±SEM for each treatment group (left panel) and by tumor weight forindividual mice in each treatment, and the mean±SEM for each treatmentgroup (right panel) in a B16F10 model.

FIG. 55 demonstrates the BLI luciferase measurements of individual miceof following treatment using control FLAG-expressing MSCs (left panel)and the combination of IL12-expressing MSCs and IL21-expressing MSCs(right panel) in a B16F10 model.

FIG. 56 shows survival curves of the treatment groups receivingIL12p70-expressing MSCs, IL21-expressing MSCs, the combination ofIL12p70 and IL21-expressing MSCs, anti-PD1, or the combination ofIL12p70 and anti-PD1.

FIG. 57A shows survival curves of mice following tumor rechallenge. FIG.57A shows naïve untreated mice.

FIG. 57B shows survival curves of mice following tumor rechallenge. FIG.57B shows mice that previously received the treatment of IL12-expressingMSCs alone.

FIG. 57C shows survival curves of mice following tumor rechallenge. FIG.57C shows mice that previously received the combination treatment ofIL12-expressing MSCs and IL21-expressing MSCs.

FIG. 58A shows dose-dependent efficacy of treatment using mMSCsengineered to express murine IL12 (p70) and murine IL21 from a singlelentiviral expression vector in a CT26 tumor model. FIG. 58A showssummarized BLI assessment of efficacy normalized day 17 vs day 7 forindividual mice in each treatment, and the mean±SEM for each treatmentgroup.

FIG. 58B shows dose-dependent efficacy of treatment using mMSCsengineered to express murine IL12 (p70) and murine IL21 from a singlelentiviral expression vector in a CT26 tumor model. FIG. 58B shows BLImeasurements over time for individual mice.

FIG. 58C shows dose-dependent efficacy of treatment using mMSCsengineered to express murine IL12 (p70) and murine IL21 from a singlelentiviral expression vector in a CT26 tumor model. FIG. 58C shows BLImeasurements over time for individual mice.

FIG. 58D shows dose-dependent efficacy of treatment using mMSCsengineered to express murine IL12 (p70) and murine IL21 from a singlelentiviral expression vector in a CT26 tumor model. FIG. 58D showssurvival curves of the treatment groups.

FIG. 59A shows dose-dependent efficacy of treatment using mMSCsengineered to express murine IL12 (p70) and murine IL21 from a singlelentiviral expression vector in a B16F10 tumor model. FIG. 59A showssummarized BLI assessment of efficacy normalized day 17 vs day 7 forindividual mice in each treatment, and the mean±SEM for each treatmentgroup.

FIG. 59B shows efficacy of treatment using control FLAG mMSCs (right) orPBS (left) in a B16F10 tumor model. FIG. 59B shows BLI measurements overtime for individual mice.

FIG. 59C shows dose-dependent efficacy of treatment using mMSCsengineered to express murine IL12 (p70) and murine IL21 from a singlelentiviral expression vector in a B16F10 tumor model. FIG. 59C shows BLImeasurements over time for individual mice.

FIG. 59D shows dose-dependent efficacy of treatment using mMSCsengineered to express murine IL12 (p70) and murine IL21 from a singlelentiviral expression vector in a B16F10 tumor model. FIG. 59D shows BLImeasurements over time for individual mice for multiple administrationsof higher doses.

FIG. 59E shows dose-dependent efficacy of treatment using mMSCsengineered to express murine IL12 (p70) and murine IL21 from a singlelentiviral expression vector in a B16F10 tumor model. FIG. 59E showssurvival curves of the treatment groups.

FIG. 60A shows dose-dependent efficacy of treatment using mMSCsengineered to express murine IL12 (p70) and murine IL21 from a singlelentiviral expression vector in a MC-38 tumor model. FIG. 60A showssummarized BLI assessment of efficacy normalized day 18 vs day 9 forindividual mice in each treatment, and the mean±SEM for each treatmentgroup.

FIG. 60B shows dose-dependent efficacy of treatment using mMSCsengineered to express murine IL12 (p70) and murine IL21 from a singlelentiviral expression vector in a MC-38 tumor model. FIG. 60B shows BLImeasurements over time for individual mice.

FIG. 60C shows dose-dependent efficacy of treatment using mMSCsengineered to express murine IL12 (p70) and murine IL21 from a singlelentiviral expression vector in a MC-38 tumor model. FIG. 60C showssurvival curves of the treatment groups.

FIG. 61A shows preferential homing of human MSCs. FIG. 61A showssummarized luciferase quantification.

FIG. 61B shows preferential homing of human MSCs. FIG. 61B showsrepresentative images of luciferase signal in organs.

FIG. 62A shows production of human IL12 (left panel) and human IL21(right panel) in the peritoneal fluid (left column for each respectivetime point) and serum (right column for each respective time point) in aOVCAR8 model for individual mice in each treatment, and the mean±SEM foreach treatment group.

FIG. 62B shows transient production of murine IL12 (left panel) andmurine IL21 (right panel) in the peritoneal fluid (left column for eachrespective time point) and serum (right column for each respective timepoint) in a CT26 model for individual mice in each treatment, and themean±SEM for each treatment group.

FIG. 63A shows efficacy of mice either treated with MSCs engineered toproduce cytokines or treated with recombinant cytokine therapy in a CT26model. FIG. 63A shows survival curves of MSC-IL12 vs rIL12.

FIG. 63B shows efficacy of mice either treated with MSCs engineered toproduce cytokines or treated with recombinant cytokine therapy in a CT26model. FIG. 63B shows survival curves of MSC-IL21 vs rIL21.

FIG. 63C shows efficacy of mice either treated with MSCs engineered toproduce cytokines or treated with recombinant cytokine therapy in a CT26model. FIG. 63C shows survival curves of MSC-IL12/IL21 vs rIL12+rIL21.

FIG. 63D shows efficacy of mice either treated with MSCs engineered toproduce cytokines or treated with recombinant cytokine therapy in a CT26model. FIG. 63D shows BLI assessments of tumor burden for mice eithertreated with MSCs engineered to produce cytokines or treated withrecombinant cytokine therapy.

FIG. 63E shows efficacy of mice either treated with MSCs engineered toproduce cytokines or treated with recombinant cytokine therapy in a CT26model. FIG. 63E shows BLI assessments of tumor burden for mice eithertreated with MSCs engineered to produce cytokines or treated withrecombinant cytokine therapy.

FIG. 64A shows efficacy of mice either treated with MSCs engineered toproduce cytokines or treated with recombinant cytokine therapy in aB16F10 model. FIG. 64A shows tumor weight assessments of tumor burdenfor individual mice in each treatment, and the mean±SEM for eachtreatment group, for mice either treated with MSCs engineered to producecytokines or treated with recombinant cytokine therapy.

FIG. 64B shows efficacy of mice either treated with MSCs engineered toproduce cytokines or treated with recombinant cytokine therapy in aB16F10 model. FIG. 64B shows survival curves of treatment groups.

FIG. 65A shows the immune profile of mice following treatment with MSCsproducing both IL12 and IL21 in a CT26 IP tumor model. Results shown aremulticolor flow cytometry analysis used to characterize immuneinfiltrates in response to treatment. FIG. 65A shows T-cell subsets andactivation markers (CD3, CD4, CD8, CD8/CD38+) for individual mice ineach treatment, and the mean±SEM for each treatment group.

FIG. 65B shows the immune profile of mice following treatment with MSCsproducing both IL12 and IL21 in a CT26 IP tumor model. Results shown aremulticolor flow cytometry analysis used to characterize immuneinfiltrates in response to treatment. FIG. 65B shows T-cell subsets andactivation markers (CD8/IFNg+, CD8/Gzmb+, NK/Gzmb+ and ratioCD8:Tregs-FoxP3) for individual mice in each treatment, and the mean±SEMfor each treatment group.

FIG. 65C shows the immune profile of mice following treatment with MSCsproducing both IL12 and IL21 in a CT26 IP tumor model. Results shown aremulticolor flow cytometry analysis used to characterize immuneinfiltrates in response to treatment. FIG. 65C shows the immune profileof antigen-presenting cells such as dendritic cells for individual micein each treatment, and the mean±SEM for each treatment group.

DETAILED DESCRIPTION

Mesenchymal stem cells (MSCs) (also referred to as mesenchymal stromalcells, multipotent stromal cells, marrow stromal cells, or multipotentmesenchymal stromal cells) are a subset of non-hematopoietic adult stemcells that originate from the mesoderm. They possess self-renewalability and multilineage differentiation into not only mesodermlineages, such as chondrocytes, osteocytes and adipocytes, but alsoectodermic cells and endodermic cells. MSCs, free of both ethicalconcerns and teratoma formation, are the major stem cell type used forcell therapy for treatment of both immune diseases and non-immunediseases. They can be easily isolated from the bone marrow, adiposetissue, the umbilical cord, fetal liver, muscle, and lung and can besuccessfully expanded in vitro. MSCs can be defined by cell surfacemarker phenotype including a cellular marker phenotype comprisingCD105+, CD73+, CD90+, CD45−, CD34−, CD14−; a cellular marker phenotypecomprising CD105+, CD73+, CD90+, CD11b−, CD79α−; or a cellular markerphenotype comprising CD105+, CD73+, CD90+, CD19−, HLA class II−, asdiscussed in greater detail in Dominici, et al. (Cytotherapy. 2006;8(4):315-7), incorporated by reference for all purposes. Further, whenMSCs are delivered exogenously and systemically to humans and animals,they tend to home to (migrate directly to) damaged tissue sites withinflammation, including tumor microenvironments and metastatic regions.The inflammation-directed MSC homing involves several important celltrafficking-related molecules, including chemokines, adhesion molecules,and matrix metalloproteinases (MMPs).

Provided herein are methods of engineering cells, such as MSCs, toproduce effector molecules that modulate different tumor-mediatedimmunosuppressive mechanisms. These MSCs are referred to herein as“engineered MSCs.” These MSCs, which typically contain engineerednucleic acid, do not occur in nature. In some embodiments, the MSCs areengineered to include a nucleic acid comprising a promoter operablylinked to a nucleotide sequence encoding an effector molecule, forexample, one that stimulates an immune response.

Also provided herein are methods of engineering cells such as immunecells, including, but not limited to natural killer (NK) cell, NKT cell,innate lymphoid cell, tumor-infiltrating lymphocyte (TIL), mast cell,eosinophil, basophil, monocyte, macrophage, neutrophil, myeloid cell,dendritic cell, T cell, CD8+ T cell, CD4+ T cell, cytotoxic T lymphocyte(CTL), viral-specific T cell, gamma-delta T cell, T regulatory cell, andB cell, to produce effector molecules. These cells, including both MSCsand immune cells, are referred to herein as “engineered cells.” Thesecells, which typically contain engineered nucleic acid, do not occur innature. In some embodiments, the cells are engineered to include anucleic acid comprising a promoter operably linked to a nucleotidesequence encoding an effector molecule, for example, one that stimulatesan immune response.

An “effector molecule,” refers to a molecule (e.g., a nucleic acid suchas DNA or RNA, or a protein (polypeptide) or peptide) that binds toanother molecule and modulates the biological activity of that moleculeto which it binds. For example, an effector molecule may act as a ligandto increase or decrease enzymatic activity, gene expression, or cellsignaling. Thus, in some embodiments, an effector molecule modulates(activates or inhibits) different immunomodulatory mechanisms. Bydirectly binding to and modulating a molecule, an effector molecule mayalso indirectly modulate a second, downstream molecule. In someembodiments, an effector molecule is a secreted molecule, while in otherembodiments, an effector molecule is bound to the cell surface orremains intracellular. For example, effector molecules includeintracellular transcription factors, microRNA, and shRNAs that modifythe internal cell state to, for example, enhance immunomodulatoryactivity, homing properties, or persistence of the cell. Non-limitingexamples of effector molecules include cytokines, chemokines, enzymesthat modulate metabolite levels, antibodies or decoy molecules thatmodulate cytokines, homing molecules, and/or integrins.

The term “modulate” encompasses maintenance of a biological activity,inhibition (partial or complete) of a biological activity, andstimulation/activation (partial or complete) of a biological activity.The term also encompasses decreasing or increasing (e.g., enhancing) abiological activity. Two different effector molecules are considered to“modulate different tumor-mediated immunosuppressive mechanisms” whenone effector molecule modulates a tumor-mediated immunosuppressivemechanism (e.g., stimulates T cell signaling) that is different from thetumor-mediated immunosuppressive mechanism modulated by the othereffector molecule (e.g., stimulates antigen presentation and/orprocessing).

Modulation by an effector molecule may be direct or indirect. Directmodulation occurs when an effector molecule binds to another moleculeand modulates activity of that molecule. Indirect modulation occurs whenan effector molecule binds to another molecule, modulates activity ofthat molecule, and as a result of that modulation, the activity of yetanother molecule (to which the effector molecule is not bound) ismodulated.

In some embodiments, modulation of a tumor-mediated immunosuppressivemechanism by at least one effector molecule results in an increase in animmunostimulatory and/or anti-tumor immune response (e.g., systemicallyor in the tumor microenvironment) by at least 10% (e.g., 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). For example, modulation ofa tumor-mediated immunosuppressive mechanism may result in an increasein an immunostimulatory and/or anti-tumor immune response by at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 100%. In some embodiments,modulation of a tumor-mediated immunosuppressive mechanism results in anincrease in an immunostimulatory and/or anti-tumor immune response10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%,10-200%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%,20-100%, 20-200%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, or 50-200%.It should be understood that “an increase” in an immunostimulatoryand/or anti-tumor immune response, for example, systemically or in atumor microenvironment, is relative to the immunostimulatory and/oranti-tumor immune response that would otherwise occur, in the absence ofthe effector molecule(s).

In some embodiments, modulation of a tumor-mediated immunosuppressivemechanism by at least one effector molecule results in an increase in animmunostimulatory and/or anti-tumor immune response (e.g., systemicallyor in the tumor microenvironment) by at least 2 fold (e.g., 2, 3, 4, 5,10, 25, 20, 25, 50, or 100 fold). For example, modulation of atumor-mediated immunosuppressive mechanism may result in an increase inan immunostimulatory and/or anti-tumor immune response by at least 3fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50fold, or at least 100 fold. In some embodiments, modulation of atumor-mediated immunosuppressive mechanism results in an increase in animmunostimulatory and/or anti-tumor immune response by 2-10, 2-20, 2-30,2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100 fold.

Non-limiting examples of immunostimulatory and/or anti-tumor immunemechanisms include T cell signaling, activity and/or recruitment,antigen presentation and/or processing, natural killer cell-mediatedcytotoxic signaling, activity and/or recruitment, dendritic celldifferentiation and/or maturation, immune cell recruitment,pro-inflammatory macrophage signaling, activity and/or recruitment,stroma degradation, immunostimulatory metabolite production, stimulatorof interferon genes (STING) signaling (which increases the secretion ofIFN and Th1 polarization, promoting an anti-tumor immune response),and/or Type I interferon signaling. An effector molecule may stimulateat least one (one or more) of the foregoing immunostimulatorymechanisms, thus resulting in an increase in an immunostimulatoryresponse. Changes in the foregoing immunostimulatory and/or anti-tumorimmune mechanisms may be assessed, for example, using in vitro assaysfor T cell proliferation or cytotoxicity, in vitro antigen presentationassays, expression assays (e.g., of particular markers), and/or cellsecretion assays (e.g., of cytokines).

In some embodiments, modulation of a tumor-mediated immunosuppressivemechanism by at least one effector molecule results in a decrease in animmunosuppressive response (e.g., systemically or in the tumormicroenvironment) by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, or 200%). For example, modulation of atumor-mediated immunosuppressive mechanism may result in a decrease inan immunosuppressive response by at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 100%. In some embodiments, modulation of a tumor-mediatedimmunosuppressive mechanism results in a decrease in animmunosuppressive response 10-20%, 10-30%, 10-40%, 10-50%, 10-60%,10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20-50%,20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%, 50-70%,50-80%, 50-90%, 50-100%, or 50-200%. It should be understood that “adecrease” in an immunosuppressive response, for example, systemically orin a tumor microenvironment, is relative to the immunosuppressiveresponse that would otherwise occur, in the absence of the effectormolecule(s).

In some embodiments, modulation of a tumor-mediated immunosuppressivemechanism by at least one effector molecule results in a decrease in animmunosuppressive response (e.g., systemically or in the tumormicroenvironment) by at least 2 fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25,50, or 100 fold). For example, modulation of a tumor-mediatedimmunosuppressive mechanism may result in a decrease in animmunosuppressive response by at least 3 fold, at least 5 fold, at least10 fold, at least 20 fold, at least 50 fold, or at least 100 fold. Insome embodiments, modulation of a tumor-mediated immunosuppressivemechanism results in a decrease in an immunosuppressive response by2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70, 2-80, 2-90, or 2-100 fold.

Non-limiting examples of immunosuppressive mechanisms include negativecostimulatory signaling, pro-apoptotic signaling of cytotoxic cells(e.g., T cells and/or NK cells), T regulatory (Treg) cell signaling,tumor checkpoint molecule production/maintenance, myeloid-derivedsuppressor cell signaling, activity and/or recruitment,immunosuppressive factor/metabolite production, and/or vascularendothelial growth factor signaling. An effector molecule may inhibit atleast one (one or more) of the foregoing immunosuppressive mechanisms,thus resulting in a decrease in an immunosuppressive response. Changesin the foregoing immunosuppressive mechanisms may be assessed, forexample, by assaying for an increase in T cell proliferation and/or anincrease in IFNγ production (negative co-stimulatory signaling, T_(reg)cell signaling and/or MDSC); Annexin V/PI flow staining (pro-apoptoticsignaling); flow staining for expression, e.g., PDL1 expression (tumorcheckpoint molecule production/maintenance); ELISA, LUMINEX®, RNA viaqPCR, enzymatic assays, e.g., IDO tryptophan catabolism(immunosuppressive factor/metabolite production); and phosphorylation ofPI3K, Akt, p38 (VEGF signaling).

In some embodiments, cells, such as MSCs, are engineered to expressmembrane-tethered anti-CD3 and/or anti-CD28 agonist extracellulardomains.

In some embodiments, cells, such as MSCs, are engineered to produce atleast two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) effector molecules,each of which modulates a different tumor-mediated immunosuppressivemechanism. In other embodiments, cells are engineered to produce atleast one effector molecule that is not natively produced by the cells.Such an effector molecule may, for example, complement the function ofeffector molecules natively produced by the cells

In some embodiments, effector molecules function additively: the effectof two effector molecules, for example, may be equal to the sum of theeffect of the two effector molecules functioning separately. In otherembodiments, effector molecules function synergistically: the effect oftwo effector molecules, for example, may be greater than the combinedfunction of the two effector molecules. The present disclosure alsoencompasses additivity and synergy between an effector molecule(s) andthe immune cell (e.g., MSC) from which they are produced.

Effector molecules that modulate tumor-mediated immunosuppressivemechanisms and/or modify tumor microenvironments may be, for example,secreted factors (e.g., cytokines, chemokines, antibodies, and/or decoyreceptors that modulate extracellular mechanisms involved in the immunesystem), inhibitors (e.g., antibodies, antibody fragments, ligand TRAPand/or small blocking peptides), intracellular factors that control cellstate (e.g., microRNAs and/or transcription factors that modulate thestate of cells to enhance pro-inflammatory properties), factors packagedinto exosomes (e.g., microRNAs, cytosolic factors, and/or extracellularfactors), surface displayed factors (e.g., checkpoint inhibitors,TRAIL), and and/or metabolic genes (e.g., enzymes that produce/modulateor degrade metabolites or amino acids).

In some embodiments, effector molecules may be selected from thefollowing non-limiting classes of molecules: cytokines, antibodies,chemokines, nucleotides, peptides, and enzymes. Non-limiting examples ofthe foregoing classes of effector molecules are listed in Table 1 andspecific sequences encoding exemplary effector molecules are listed inTable 6. Effector molecules can be human, such as those listed in Table1 or Table 6 or human equivalents of murine effector molecules listed inTable 1 or Table 6. Effector molecules can be human-derived, such as theendogenous human effector molecule or an effector molecule modifiedand/or optimized for function, e.g., codon optimized to improveexpression, modified to improve stability, or modified at its signalsequence (see below). Various programs and algorithms for optimizingfunction are known to those skilled in the art and can be selected basedon the improvement desired, such as codon optimization for a specificspecies (e.g., human, mouse, bacteria, etc.).

TABLE 1 Exemplary Effector Molecules Effector name Category Functionanti-CD40 or CD40 Agonist antibody Stimulates B-cells and antigen Ligandpresenting cells. Flt3L Ligand agonist Stimulates myeloid cells andantigen presenting cells CXCL10-11 fusion Chemokine Attracts T-cellsTGFb blocking Antagonist Inhibit TGFb pathway, TME peptides peptidesmodifier Adenosine TME modifier Degradation of suppressive deaminase(ADA) adenosine in the TME Kyneurinase TME modifier Degradation ofkyneurine HPGE2 TME modifier Degradation of PGE2 CXCL13 ChemokineAttracts B-cells anti PD-1/PD-L1 Agonist antibody Remove checkpointanti-CTLA-4 Agonist antibody Remove checkpoint anti-VEGF AntagonistNeutralizes an antibody immunosuppressive/angiogenesis factor anti-TNFaAntagonist Neutralizes cytokine/pro-tumor antibody factor anti-IL-10Antagonist Neutralizes immunosuppressive antibody cytokine anti-Antagonist Neutralizes pro-tumor chemokine SDF1/CXCL12 antibody (TβRII)2trap Capture trap Neutralizes an immunosuppressive cytokine CCL21Chemokine Attracts leukocytes/NK CCL1 Chemokine Attracts leukocytes/NKCCL17 Chemokine Attracts leukocytes/NK CCL19 Chemokine Attractsleukocytes/NK CCL21 Chemokine Attracts leukocytes/NK CCL20 ChemokineAttracts leukocytes/NK CCL21a Chemokine Attracts leukocytes/NK MIP1b(CCL5) Chemokine Attracts leukocytes/NK CXCL10 Chemokine Attractsleukocytes/NK CXCL11 Chemokine Attracts leukocytes/NK CCL2 ChemokineAttracts monocytes MIP-1alpha (CCL3) Chemokine Attracts leukocytes/NKXCL1 Chemokine Attracts leukocytes/NK IFNbeta Cytokine T cell response,tumor cell killing IFNgamma Cytokine T cell response, tumor cell killingIL-12 Cytokine T cells, NK cells IL-1beta Cytokine T cells, NK cellsIL-15 Cytokine Stimulates T-cells and NK IL-2 Cytokine StimulatesT-cells and NK IL-21 Cytokine Stimulates T-cells IL-24 CytokineStimulates T-cells IL36-gamma Cytokine Stimulates T-cells IL-7 CytokineStimulates T-cells IL-22 Cytokine Stimulates T-cells IL-18 CytokineStimulates T-cells Granzymes/Perforin Enzyme Direct tumor cell killingOX86 (anti-OX40) ligand Stimulates T-cells anti-TGFbeta NeutralizingNeutralizes an antibody Immunosuppressive cytokine TRAIL Receptor/ligandDirect tumor cell killing FASL (CD49L) Receptor/ligand Direct tumor cellkilling OX40-L Receptor/Ligand Stimulates T-cells cGAS secreted moleculeStimulates antigen-presenting cells 41BBL secreted moleculeCo-activation of T-cells CD40L secreted molecule Stimulates T-cellsGM-CSF secreted molecule Growth factor for monocytes STING secretedmolecule Stimulates antigen-presenting cells HAC-V Antagonist inhibitscheckpoint ‘microbody’_PD1 antibody yCD Pro-drug Converts to cytotoxicmolecule upon activation CpG/Nucleotides Nucleotides STING agonist

In some embodiments, cells, such as MSCs, comprise an engineered nucleicacid that comprises a promoter operably linked to a nucleotide sequenceencoding an effector molecule. In some embodiments, an engineerednucleic acid comprises a promoter operably linked to a nucleotidesequence encoding at least 2 effector molecules. For example, theengineered nucleic acid may comprise a promoter operably linked to anucleotide sequence encoding at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 8, at least 9, or at least 10effector molecules. In some embodiments, an engineered nucleic acidcomprises a promoter operably linked to a nucleotide sequence encoding1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more effector molecules.

Engineered cells, such as engineered MSCs, in some embodiments, areengineered to include at least two engineered nucleic acids, eachcomprising a promoter operably linked to a nucleotide sequence encodingat least one (e.g., 1, 2 or 3) effector molecule. For example, the cellsmay be engineered to comprise at least 2, at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 8, at least 9, orat least 10, engineered nucleic acids, each comprising a promoteroperably linked to a nucleotide sequence encoding at least one (e.g., 1,2 or 3) effector molecule. In some embodiments, the cells are engineeredto comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more engineered nucleicacids, each comprising a promoter operably linked to a nucleotidesequence encoding at least one (e.g., 1, 2 or 3) effector molecule.

An “engineered nucleic acid” is a nucleic acid that does not occur innature. It should be understood, however, that while an engineerednucleic acid as a whole is not naturally-occurring, it may includenucleotide sequences that occur in nature. In some embodiments, anengineered nucleic acid comprises nucleotide sequences from differentorganisms (e.g., from different species). For example, in someembodiments, an engineered nucleic acid includes a murine nucleotidesequence, a bacterial nucleotide sequence, a human nucleotide sequence,and/or a viral nucleotide sequence. The term “engineered nucleic acids”includes recombinant nucleic acids and synthetic nucleic acids. A“recombinant nucleic acid” refers to a molecule that is constructed byjoining nucleic acid molecules and, in some embodiments, can replicatein a live cell. A “synthetic nucleic acid” refers to a molecule that isamplified or chemically, or by other means, synthesized. Syntheticnucleic acids include those that are chemically modified, or otherwisemodified, but can base pair with naturally-occurring nucleic acidmolecules. Recombinant nucleic acids and synthetic nucleic acids alsoinclude those molecules that result from the replication of either ofthe foregoing. Engineered nucleic acid of the present disclosure may beencoded by a single molecule (e.g., included in the same plasmid orother vector) or by multiple different molecules (e.g., multipledifferent independently-replicating molecules).

Engineered nucleic acid of the present disclosure may be produced usingstandard molecular biology methods (see, e.g., Green and Sambrook,Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press).In some embodiments, engineered nucleic acid constructs are producedusing GIBSON ASSEMBLY® Cloning (see, e.g., Gibson, D. G. et al. NatureMethods, 343-345, 2009; and Gibson, D. G. et al. Nature Methods,901-903, 2010, each of which is incorporated by reference herein).GIBSON ASSEMBLY® typically uses three enzymatic activities in asingle-tube reaction: 5′ exonuclease, the ‘Y extension activity of a DNApolymerase and DNA ligase activity. The 5′ exonuclease activity chewsback the 5′ end sequences and exposes the complementary sequence forannealing. The polymerase activity then fills in the gaps on theannealed regions. A DNA ligase then seals the nick and covalently linksthe DNA fragments together. The overlapping sequence of adjoiningfragments is much longer than those used in Golden Gate Assembly, andtherefore results in a higher percentage of correct assemblies. In someembodiments, engineered nucleic acid constructs are produced usingIN-FUSION® cloning (Clontech).

A “promoter” refers to a control region of a nucleic acid sequence atwhich initiation and rate of transcription of the remainder of a nucleicacid sequence are controlled. A promoter may also contain sub-regions atwhich regulatory proteins and molecules may bind, such as RNA polymeraseand other transcription factors. Promoters may be constitutive,inducible, repressible, tissue-specific or any combination thereof. Apromoter drives expression or drives transcription of the nucleic acidsequence that it regulates. Herein, a promoter is considered to be“operably linked” when it is in a correct functional location andorientation in relation to a nucleic acid sequence it regulates tocontrol (“drive”) transcriptional initiation and/or expression of thatsequence.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment of a given gene or sequence. Such apromoter can be referred to as “endogenous.” In some embodiments, acoding nucleic acid sequence may be positioned under the control of arecombinant or heterologous promoter, which refers to a promoter that isnot normally associated with the encoded sequence in its naturalenvironment. Such promoters may include promoters of other genes;promoters isolated from any other cell; and synthetic promoters orenhancers that are not “naturally occurring” such as, for example, thosethat contain different elements of different transcriptional regulatoryregions and/or mutations that alter expression through methods ofgenetic engineering that are known in the art. In addition to producingnucleic acid sequences of promoters and enhancers synthetically,sequences may be produced using recombinant cloning and/or nucleic acidamplification technology, including polymerase chain reaction (PCR)(see, e.g., U.S. Pat. Nos. 4,683,202 and 5,928,906).

Promoters of an engineered nucleic acid may be “inducible promoters,”which refer to promoters that are characterized by regulating (e.g.,initiating or activating) transcriptional activity when in the presenceof, influenced by or contacted by a signal. The signal may be endogenousor a normally exogenous condition (e.g., light), compound (e.g.,chemical or non-chemical compound) or protein (e.g., cytokine) thatcontacts an inducible promoter in such a way as to be active inregulating transcriptional activity from the inducible promoter.Activation of transcription may involve directly acting on a promoter todrive transcription or indirectly acting on a promoter by inactivation arepressor that is preventing the promoter from driving transcription.Conversely, deactivation of transcription may involve directly acting ona promoter to prevent transcription or indirectly acting on a promoterby activating a repressor that then acts on the promoter.

A promoter is “responsive to” or “modulated by” a local tumor state(e.g., inflammation or hypoxia) or signal if in the presence of thatstate or signal, transcription from the promoter is activated,deactivated, increased, or decreased. In some embodiments, the promotercomprises a response element. A “response element” is a short sequenceof DNA within a promoter region that binds specific molecules (e.g.,transcription factors) that modulate (regulate) gene expression from thepromoter. Response elements that may be used in accordance with thepresent disclosure include, without limitation, a phloretin-adjustablecontrol element (PEACE), a zinc-finger DNA-binding domain (DBD), aninterferon-gamma-activated sequence (GAS) (Decker, T. et al. JInterferon Cytokine Res. 1997 March; 17(3):121-34, incorporated hereinby reference), an interferon-stimulated response element (ISRE) (Han, K.J. et al. J Biol Chem. 2004 Apr. 9; 279(15):15652-61, incorporatedherein by reference), a NF-kappaB response element (Wang, V. et al. CellReports. 2012; 2(4): 824-839, incorporated herein by reference), and aSTAT3 response element (Zhang, D. et al. J of Biol Chem. 1996; 271:9503-9509, incorporated herein by reference). Other response elementsare encompassed herein. Response elements can also contain tandemrepeats (e.g., consecutive repeats of the same nucleotide sequenceencoding the response element) to generally increase sensitivity of theresponse element to its cognate binding molecule. Tandem repeats can belabeled 2×, 3×, 4×, 5×, etc. to denote the number of repeats present.

Non-limiting examples of responsive promoters (also referred to as“inducible promoters”) (e.g., TGF-beta responsive promoters) are listedin Table 2, which shows the design of the promoter and transcriptionfactor, as well as the effect of the inducer molecule towards thetranscription factor (TF) and transgene transcription (T) is shown (B,binding; D, dissociation; n.d., not determined) (A, activation; DA,deactivation; DR, derepression) (see Horner, M. & Weber, W. FEBS Letters586 (2012) 20784-2096m, and references cited therein). Othernon-limiting examples of inducible promoters include those presented inTable 3.

TABLE 2 Examples of Responsive Promoters. Response to Transcriptioninducer System Promoter and operator factor (TF) Inducer molecule TF TTranscriptional activator-responsive promoters AIR PAIR (OalcA- AlcRAcetaldehyde n.d. A PhCMVmin) ART PART (OARG- ArgR-VP16 1-Arginine B APhCMVmin) BIT PBIT3 (OBirA3- BIT (BirA-VP16) Biotin B A PhCMVmin)Cumate- PCR5 (OCuO6- cTA (CymR- Cumate D DA activator PhCMVmin) VP16)Cumate- PCR5 (OCuO6- rcTA (rCymR- Cumate B A reverse PhCMVmin) VP16)activator E-OFF PETR (OETR- ET (E-VP16) Erythromycin D DA PhCMVmin)NICE-OFF PNIC (ONIC- NT (HdnoR- 6-Hydroxy-nicotine D DA PhCMVmin) VP16)PEACE PTtgR1 (OTtgR- TtgA1 (TtgR- Phloretin D DA PhCMVmin) VP16) PIP-OFFPPIR (OPIR-Phsp70min) PIT (PIP-VP16) Pristinamycin I D DA QuoRex PSCA(OscbR- SCA (ScbR- SCB1 D DA PhCMVmin)PSPA VP16) (OpapRI-PhCMVmin) RedoxPROP (OROP- REDOX (REX- NADH D DA PhCMVmin) VP16) TET-OFF PhCMV*-1(OtetO7- tTA (TetR- Tetracycline D DA PhCMVmin) VP16) TET-ON PhCMV*-1(OtetO7- rtTA (rTetR- Doxycycline B A PhCMVmin) VP16) TIGR PCTA (OrheO-CTA (RheA- Heat D DA PhCMVmin) VP16) TraR O7x(tra box)-PhCMVmin p65-TraR3-Oxo-C8-HSL B A VAC-OFF P1VanO2 (OVanO2- VanA1 (VanR- Vanillic acid DDA PhCMVmin) VP16) Transcriptional repressor-responsive promotersCumate- PCuO (PCMV5-OCuO) CymR Cumate D DR repressor E-ON PETRON8(PSV40- E-KRAB Erythromycin D DR OETR8) NICE-ON PNIC (PSV40-ONIC8) NS(HdnoR- 6-Hydroxy-nicotine D DR KRAB) PIP-ON PPIRON (PSV40-OPIR3) PIT3(PIP- Pristinamycin I D DR KRAB) Q-ON PSCAON8 (PSV40- SCS (ScbR- SCB1 DDR OscbR8) KRAB) TET- OtetO-PHPRT tTS-H4 (TetR- Doxycycline D DRON<comma> HDAC4) repressor-based T-REX PTetO (PhCMV- TetR Tetracycline DDR OtetO2) UREX PUREX8 (PSV40- mUTS (KRAB- Uric acid D DR OhucO8) HucR)VAC-ON PVanON8 (PhCMV- VanA4 (VanR- Vanillic acid D DR OVanO8) KRAB)Hybrid promoters QuoRexPIP- OscbR8-OPIR3- SCAPIT3 SCB1Pristinamycin I DDDADR ON(NOT IF gate) PhCMVmin QuoRexE- OscbR-OETR8- SCAE-KRABSCB1Erythromycin DD DADR ON(NOT IF gate) PhCMVmin TET-OFFE-OtetO7-OETR8- tTAE-KRAB TetracyclineEry- DD DADR ON(NQT IF gate)PhCMVmin thromycin TET-OFFPIP- OtetO7-OPIR3- tTAPIT3E-TetracyclinePristina- DDD DADRDR ONE-ON OETR8- KRAB mycin IErythromycinPhCMVmin

TABLE 3 Exemplary Inducible Promoters Name DNA SEQUENCE Sourceminimal promoter; AGAGGGTATATAATGGAAGCTCGACTT EU581860.1 minPCCAG (SEQ ID NO: 1) (Promega) NFkB response elementGGGAATTTCCGGGGACTTTCCGGGAAT EU581860.1 protein promoter; 5xTTCCGGGGACTTTCCGGGAATTTCC (Promega) NFkB-RE (SEQ ID NO: 2)CREB response element CACCAGACAGTGACGTCAGCTGCCAGA DQ904461.1protein promoter; TCCCATGGCCGTCATACTGTGACGTCTT (Promega) 4x CRETCAGACACCCCATTGACGTCAATGGGA GAA (SEQ ID NO: 3) NFAT response elementGGAGGAAAAACTGTTTCATACAGAAGG DQ904462.1 protein promoter; 3xCGTGGAGGAAAAACTGTTTCATACAGA (Promega) NFAT binding sitesAGGCGTGGAGGAAAAACTGTTTCATAC AGAAGGCGT (SEQ ID NO: 4)SRF response element AGGATGTCCATATTAGGACATCTAGGA FJ773212.1protein promoter; TGTCCATATTAGGACATCTAGGATGTC (Promega) 5x SRECATATTAGGACATCTAGGATGTCCATA TTAGGACATCTAGGATGTCCATATTAGGACATCT (SEQ ID NO: 5) SRF response element AGTATGTCCATATTAGGACATCTACCAFJ773213.1 protein promoter 2; TGTCCATATTAGGACATCTACTATGTCC (Promega)5x SRF-RE ATATTAGGACATCTTGTATGTCCATATT AGGACATCTAAAATGTCCATATTAGGACATCT (SEQ ID NO: 6) AP1 response element TGAGTCAGTGACTCAGTGAGTCAGTGAJQ858516.1 protein promoter; 6x CTCAGTGAGTCAGTGACTCAG (SEQ ID (Promega)API-RE NO: 7) TCF-LEF response AGATCAAAGGGTTTAAGATCAAAGGGC JX099537.1element protein TTAAGATCAAAGGGTATAAGATCAAAG (Promega) promoter;GGCCTAAGATCAAAGGGACTAAGATCA 8x TCF-LEF-RE AAGGGTTTAAGATCAAAGGGCTTAAGATCAAAGGGCCTA (SEQ ID NO: 8) SBEx4 GTCTAGACGTCTAGACGTCTAGACGTCAddgene Cat No: 16495 TAGAC (SEQ ID NO: 9) SMAD2/3-CAGACA x4CAGACACAGACACAGACACAGACA Jonk et al. (J Biol Chem. (SEQ ID NO: 10) 1998August 14;273(33):21145-52. STAT3 binding siteGgatccggtactcgagatctgcgatctaagtaagcttggcattc Addgene Sequencing Resultcggtactgttggtaaagccac (SEQ ID NO: 11) #211335

Other non-limiting examples of promoters include the cytomegalovirus(CMV) promoter, the elongation factor 1-alpha (EF1a) promoter, theelongation factor (EFS) promoter, the MND promoter (a synthetic promoterthat contains the U3 region of a modified MoMuLV LTR withmyeloproliferative sarcoma virus enhancer), the phosphoglycerate kinase(PGK) promoter, the spleen focus-forming virus (SFFV) promoter, thesimian virus 40 (SV40) promoter, and the ubiquitin C (UbC) promoter (seeTable 4).

TABLE 4 Exemplary Constitutive Promoters Name DNA SEQUENCE CMVGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTC (SEQ ID NO: 12) EF1aGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGCCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGACCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGTCCTCGCGCCGCCGTGTATCGCCCCGCCCCGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGTCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA (SEQ ID NO: 13) EFSGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTAC (SEQ ID NO: 14) MNDTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGTTGGAACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCA (SEQ ID NO: 15) PGKGGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCGGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAG (SEQ ID NO: 16) SFFVGTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGCCC GGG (SEQ ID NO: 17)SV40 CTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCT (SEQ ID NO: 18) UbCGCGCCGGGTTTTGGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGAAGGGCGCAGGAGCGTTCCTGATCCTTCCGCCCGGACGCTCAGGACAGCGGCCCGCTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTTTAGGACGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCGAGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTGAACGCCGATGATTATATAAGGACGCGCCGGGTGTGGCACAGCTAGTTCCGTCGCAGCCGGGATTTGGGTCGCGGTTCTTGTTTGTGGATCGCTGTGATCGTCACTTGGTGAGTTGCGGGCTGCTGGGCTGGCCGGGGCTTTCGTGGCCGCCGGGCCGCTCGGTGGGACGGAAGCGTGTGGAGAGACCGCCAAGGGCTGTAGTCTGGGTCCGCGAGCAAGGTTGCCCTGAACTGGGGGTTGGGGGGAGCGCACAAAATGGCGGCTGTTCCCGAGTCTTGAATGGAAGACGCTTGTAAGGCGGGCTGTGAGGTCGTTGAAACAAGGTGGGGGGCATGGTGGGCGGCAAGAACCCAAGGTCTTGAGGCCTTCGCTAATGCGGGAAAGCTCTTATTCGGGTGAGATGGGCTGGGGCACCATCTGGGGACCCTGACGTGAAGTTTGTCACTGACTGGAGAACTCGGGTTTGTCGTCTGGTTGCGGGGGCGGCAGTTATGCGGTGCCGTTGGGCAGTGCACCCGTACCTTTGGGAGCGCGCGCCTCGTCGTGTCGTGACGTCACCCGTTCTGTTGGCTTATAATGCAGGGTGGGGCCACCTGCCGGTAGGTGTGCGGTAGGCTTTTCTCCGTCGCAGGACGCAGGGTTCGGGCCTAGGGTAGGCTCTCCTGAATCGACAGGCGCCGGACCTCTGGTGAGGGGAGGGATAAGTGAGGCGTCAGTTTCTTTGGTCGGTTTTATGTACCTATCTTCTTAAGTAGCTGAAGCTCCGGTTTTGAACTATGCGCTCGGGGTTGGCGAGTGTGTTTTGTGAAGTTTTTTAGGCACCTTTTGAAATGTAATCATTTGGGTCAATATGTAATTTTCAGTGTTAGACTAGTAAAGCTTCTGCAGGTCGACTCTAGAAAATTGTCCGCTAAATTCTGGCCGTTTTTGGCTTTTTTGTTAGAC (SEQ ID NO: 19) hEF1aV1GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGTCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCG TGA (SEQ ID NO: 20)hCAGG ACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTC (SEQ ID NO: 21) hEF1aV2Gggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgaggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacag(SEQ ID NO: 22) hACTbCCACTAGTTCCATGTCCTTATATGGACTCATCTTTGCCTATTGCGACACACACTCAATGAACACCTACTACGCGCTGCAAAGAGCCCCGCAGGCCTGAGGTGCCCCCACCTCACCACTCTTCCTATTTTTGTGTAAAAATCCAGCTTCTTGTCACCACCTCCAAGGAGGGGGAGGAGGAGGAAGGCAGGTTCCTCTAGGCTGAGCCGAATGCCCCTCTGTGGTCCCACGCCACTGATCGCTGCATGCCCACCACCTGGGTACACACAGTCTGTGATTCCCGGAGCAGAACGGACCCTGCCCACCCGGTCTTGTGTGCTACTCAGTGGACAGACCCAAGGCAAGAAAGGGTGACAAGGACAGGGTCTTCCCAGGCTGGCTTTGAGTTCCTAGCACCGCCCCGCCCCCAATCCTCTGTGGCACATGGAGTCTTGGTCCCCAGAGTCCCCCAGCGGCCTCCAGATGGTCTGGGAGGGCAGTTCAGCTGTGGCTGCGCATAGCAGACATACAACGGACGGTGGGCCCAGACCCAGGCTGTGTAGACCCAGCCCCCCCGCCCCGCAGTGCCTAGGTCACCCACTAACGCCCCAGGCCTGGTCTTGGCTGGGCGTGACTGTTACCCTCAAAAGCAGGCAGCTCCAGGGTAAAAGGTGCCCTGCCCTGTAGAGCCCACCTTCCTTCCCAGGGCTGCGGCTGGGTAGGTTTGTAGCCTTCATCACGGGCCACCTCCAGCCACTGGACCGCTGGCCCCTGCCCTGTCCTGGGGAGTGTGGTCCTGCGACTTCTAAGTGGCCGCAAGCCACCTGACTCCCCCAACACCACACTCTACCTCTCAAGCCCAGGTCTCTCCCTAGTGACCCACCCAGCACATTTAGCTAGCTGAGCCCCACAGCCAGAGGTCCTCAGGCCCTGCTTTCAGGGCAGTTGCTCTGAAGTCGGCAAGGGGGAGTGACTGCCTGGCCACTCCATGCCCTCCAAGAGCTCCTTCTGCAGGAGCGTACAGAACCCAGGGCCCTGGCACCCGTGCAGACCCTGGCCCACCCCACCTGGGCGCTCAGTGCCCAAGAGATGTCCACACCTAGGATGTCCCGCGGTGGGTGGGGGGCCCGAGAGACGGGCAGGCCGGGGGCAGGCCTGGCCATGCGGGGCCGAACCGGGCACTGCCCAGCGTGGGGCGCGGGGGCCACGGCGCGCGCCCCCAGCCCCCGGGCCCAGCACCCCAAGGCGGCCAACGCCAAAACTCTCCCTCCTCCTCTTCCTCAATCTCGCTCTCGCTCTTTTTTTTTTTCGCAAAAGGAGGGGAGAGGGGGTAAAAAAATGCTGCACTGTGCGGCGAAGCCGGTGAGTGAGCGGCGCGGGGCCAATCAGCGTGCGCCGTTCCGAAAGTTGCCTTTTATGGCTCGAGCGGCCGCGGCGGCGCCCTATAAAACCCAGCGGCGCGACGCGCCACCACCGCCGAGACCGCGTCCGCCCCGCGAGCACAGAGCCTCGCCTTTGCCGATCCGCCGCCCGTCCACACCCGCCGCCAGgtaagcccggccagccgaccggggcaggcggctcacggcccggccgcaggcggccgcggccccttcgcccgtgcagagccgccgtctgggccgcagcggggggcgcatggggggggaaccggaccgccgtggggggcgcgggagaagcccctgggcctccggagatgggggacaccccacgccagttcggaggcgcgaggccgcgctcgggaggcgcgctccgggggtgccgctctcggggcgggggcaaccggcggggtctttgtctgagccgggctcttgccaatggggatcgcagggtgggcgcggcggagcccccgccaggcccggtgggggctggggcgccattgcgcgtgcgcgctggtcctttgggcgctaactgcgtgcgcgctgggaattggcgctaattgcgcgtgcgcgctgggactcaaggcgctaactgcgcgtgcgttctggggcccggggtgccgcggcctgggctggggcgaaggcgggctcggccggaaggggtggggtcgccgcggctcccgggcgcttgcgcgcacttcctgcccgagccgctggccgcccgagggtgtggccgctgcgtgcgcgcgcgccgacccggcgctgtttgaaccgggcggaggcggggctggcgcccggttgggagggggttggggcctggcttcctgccgcgcgccgcggggacgcctccgaccagtgtttgccttttatggtaataacgcggccggcccggcttcctttgtccccaatctgggcgcgcgccggcgccccctggcggcctaaggactcggcgcgccggaagtggccagggcgggggcgacctcggctcacagcgcgcccggctat (SEQ ID NO: 23) heIF4A1GTTGATTTCCTTCATCCCTGGCACACGTCCAGGCAGTGTCGAATCCATCTCTGCTACAGGGGAAAACAAATAACATTTGAGTCCAGTGGAGACCGGGAGCAGAAGTAAAGGGAAGTGATAACCCCCAGAGCCCGGAAGCCTCTGGAGGCTGAGACCTCGCCCCCCTTGCGTGATAGGGCCTACGGAGCCACATGACCAAGGCACTGTCGCCTCCGCACGTGTGAGAGTGCAGGGCCCCAAGATGGCTGCCAGGCCTCGAGGCCTGACTCTTCTATGTCACTTCCGTACCGGCGAGAAAGGCGGGCCCTCCAGCCAATGAGGCTGCGGGGCGGGCCTTCACCTTGATAGGCACTCGAGTTATCCAATGGTGCCTGCGGGCCGGAGCGACTAGGAACTAACGTCATGCCGAGTTGCTGAGCGCCGGCAGGCGGGGCCGGGGCGGCCAAACCAATGCGATGGCCGGGGCGGAGTCGGGCGCTCTATAAGTTGTCGATAGGCGGGCACTCCGCCCTAGTTTCTAAGGACCATG (SEQ ID NO: 24) hGAPDHAGTTCCCCAACTTTCCCGCCTCTCAGCCTTTGAAAGAAAGAAAGGGGAGGGGGCAGGCCGCGTGCAGTCGCGAGCGGTGCTGGGCTCCGGCTCCAATTCCCCATCTCAGTCGCTCCCAAAGTCCTTCTGTTTCATCCAAGCGTGTAAGGGTCCCCGTCCTTGACTCCCTAGTGTCCTGCTGCCCACAGTCCAGTCCTGGGAACCAGCACCGATCACCTCCCATCGGGCCAATCTCAGTCCCTTCCCCCCTACGTCGGGGCCCACACGCTCGGTGCGTGCCCAGTTGAACCAGGCGGCTGCGGAAAAAAAAAAGCGGGGAGAAAGTAGGGCCCGGCTACTAGCGGTTTTACGGGCGCACGTAGCTCAGGCCTCAAGACCTTGGGCTGGGACTGGCTGAGCCTGGCGGGAGGCGGGGTCCGAGTCACCGCCTGCCGCCGCGCCCCCGGTTTCTATAAATTGAGCCCGCAGCCTCCCGCTTCGCTCTCTGCTCCTCCTGTTCGACAGTCAGCCGCATCTTCTTTTGCGTCGCCAGgtgaagacgggcggagagaaacccgggaggctagggacggcctgaaggcggcaggggcgggcgcaggccggatgtgttcgcgccgctgcggggtgggcccgggcggcctccgcattgcaggggcgggcggaggacgtgatgcggcgcgggctgggcatggaggcctggtgggggaggggaggggaggcgtgggtgtcggccggggccactaggcgctcactgttctctccctccgcgcagCCGAGCCACATCGCTGAGACAC (SEQ ID NO: 25)hGRP78 AGTGCGGTTACCAGCGGAAATGCCTCGGGGTCAGAAGTCGCAGGAGAGATAGACAGCTGCTGAACCAATGGGACCAGCGGATGGGGCGGATGTTATCTACCATTGGTGAACGTTAGAAACGAATAGCAGCCAATGAATCAGCTGGGGGGGCGGAGCAGTGACGTTTATTGCGGAGGGGGCCGCTTCGAATCGGCGGCGGCCAGCTTGGTGGCCTGGGCCAATGAACGGCCTCCAACGAGCAGGGCCTTCACCAATCGGCGGCCTCCACGACGGGGCTGGGGGAGGGTATATAAGCCGAGTAGGCGACGGTGAGGTCGACGCCGGCCAAGACAGCACAGACAGATTGACCTATTGGGGTGTTTCGCGAGTGTGAGAGGGAAGCGCCGCGGCCTGTATTTCTAGACCTGCCCTTCGCCTGGTTCGTGGCGCCTTGTGACCCCGGGCCCCTGCCGCCTGCAAGTCGGAAATTGCGCTGTGCTCCTGTGCTACGGCCTGTGGCTGGACTGCCTGCTGCTGCCCAACTGGCTGGCAC (SEQ ID NO: 26) hGRP94TAGTTTCATCACCACCGCCACCCCCCCGCCCCCCCGCCATCTGAAAGGGTTCTAGGGGATTTGCAACCTCTCTCGTGTGTTTCTTCTTTCCGAGAAGCGCCGCCACACGAGAAAGCTGGCCGCGAAAGTCGTGCTGGAATCACTTCCAACGAAACCCCAGGCATAGATGGGAAAGGGTGAAGAACACGTTGCCATGGCTACCGTTTCCCCGGTCACGGAATAAACGCTCTCTAGGATCCGGAAGTAGTTCCGCCGCGACCTCTCTAAAAGGATGGATGTGTTCTCTGCTTACATTCATTGGACGTTTTCCCTTAGAGGCCAAGGCCGCCCAGGCAAAGGGGCGGTCCCACGCGTGAGGGGCCCGCGGAGCCATTTGATTGGAGAAAAGCTGCAAACCCTGACCAATCGGAAGGAGCCACGCTTCGGGCATCGGTCACCGCACCTGGACAGCTCCGATTGGTGGACTTCCGCCCCCCCTCACGAATCCTCATTGGGTGCCGTGGGTGCGTGGTGCGGCGCGATTGGTGGGTTCATGTTTCCCGTCCCCCGCCCGCGAGAAGTGGGGGTGAAAAGCGGCCCGACCTGCTTGGGGTGTAGTGGGCGGACCGCGCGGCTGGAGGTGTGAGGATCCGAACCCAGGGGTGGGGGGTGGAGGCGGCTCCTGCGATCGAAGGGGACTTGAGACTCACCGGCCGCACGTC (SEQ ID NO: 27) hHSP70GGGCCGCCCACTCCCCCTTCCTCTCAGGGTCCCTGTCCCCTCCAGTGAATCCCAGAAGACTCTGGAGAGTTCTGAGCAGGGGGCGGCACTCTGGCCTCTGATTGGTCCAAGGAAGGCTGGGGGGCAGGACGGGAGGCGAAAACCCTGGAATATTCCCGACCTGGCAGCCTCATCGAGCTCGGTGATTGGCTCAGAAGGGAAAAGGCGGGTCTCCGTGACGACTTATAAAAGCCCAGGGGCAAGCGGTCCGGATAACGGCTAGCCTGAGGAGCTGCTGCGACAGTCCACTACCTTTTTCGAGAGTGACTCCCGTTGTCCCAAGGCTTCCCAGAGCGAACCTGTGCGGCTGCAGGCACCGGCGCGTCGAGTTTCCGGCGTCCGGAAGGACCGAGCTCTTCTCGCGGATCCAGTGTTCCGTTTCCAGCCCCCAATCTCAGAGCGGAGCCGACAGAGAGCAGGGAACCC (SEQ ID NO: 28) hKINbGCCCCACCCCCGTCCGCGTTACAACCGGGAGGCCCGCTGGGTCCTGCACCGTCACCCTCCTCCCTGTGACCGCCCACCTGATACCCAAACAACTTTCTCGCCCCTCCAGTCCCCAGCTCGCCGAGCGCTTGCGGGGAGCCACCCAGCCTCAGTTTCCCCAGCCCCGGGCGGGGCGAGGGGCGATGACGTCATGCCGGCGCGCGGCATTGTGGGGCGGGGCGAGGCGGGGCGCCGGGGGGAGCAACACTGAGACGCCATTTTCGGCGGCGGGAGCGGCGCAGGCGGCCGAGCGGGACTGGCTGGGTCGGCTGGGCTGCTGGTGCGAGGAGCCGCGGGGCTGTGCTCGGCGGCCAAGGGGACAGCGCGTGGGTGGCCGAGGATGCTGCGGGGCGGTAGCTCCGGCGCCCCTCGCTGGTGACTGCTGCGCCGTGCCTCACACAGCCGAGGCGGGCTCGGCGCACAGTCGCTGCTCCGCGCTCGCGCCCGGCGGCGCTCCAGGTGCTGACAGCGCGAGAGAGCGCGGCCTCAGGAGCAACAC (SEQ ID NO: 29) hUBIbTTCCAGAGCTTTCGAGGAAGGTTTCTTCAACTCAAATTCATCCGCCTGATAATTTTCTTATATTTTCCTAAAGAAGGAAGAGAAGCGCATAGAGGAGAAGGGAAATAATTTTTTAGGAGCCTTTCTTACGGCTATGAGGAATTTGGGGCTCAGTTGAAAAGCCTAAACTGCCTCTCGGGAGGTTGGGCGCGGCGAACTACTTTCAGCGGCGCACGGAGACGGCGTCTACGTGAGGGGTGATAAGTGACGCAACACTCGTTGCATAAATTTGCGCTCCGCCAGCCCGGAGCATTTAGGGGCGGTTGGCTTTGTTGGGTGAGCTTGTTTGTGTCCCTGTGGGTGGACGTGGTTGGTGATTGGCAGGATCCTGGTATCCGCTAACAGgtactggcccacagccgtaaagacctgcgggggcgtgagaggggggaatgggtgaggtcaagctggaggcttcttggggttgggtgggccgctgaggggaggggagggcgaggtgacgcgacacccggcctttctgggagagtgggccttgttgacctaaggggggcgagggcagttggcacgcgcacgcgccgacagaaactaacagacattaaccaacagcgattccgtcgcgtttacttgggaggaaggcggaaaagaggtagtttgtgtggcttctggaaaccctaaatttggaatcccagtatgagaatggtgtcccttcttgtgtttcaatgggatttttacttcgcgagtcttgtgggtttggttttgttttcagtttgcctaacaccgtgcttaggtttgaggcagattggagttcggtcgggggagtttgaatatccggaacagttagtggggaaagctgtggacgcttggtaagagagcgctctggattttccgctgttgacgttgaaaccttgaatgacgaatttcgtattaagtgacttagccttgtaaaattgaggggaggcttgcggaatattaacgtatttaaggcattttgaaggaatagttgctaattttgaagaatattaggtgtaaaagcaagaaatacaatgatcctgaggtgacacgcttatgttttacttttaaactagGTCACC (SEQ ID NO: 30)

In some embodiments, a promoter of the present disclosure is modulatedby signals within a tumor microenvironment. A tumor microenvironment isconsidered to modulate a promoter if, in the presence of the tumormicroenvironment, the activity of the promoter is increased or decreasedby at least 10%, relative to activity of the promoter in the absence ofthe tumor microenvironment. In some embodiments, the activity of thepromoter is increased or decreased by at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 100%, relative to activity of the promoter in theabsence of the tumor microenvironment. For example, the activity of thepromoter is increased or decreased by 10-20%, 10-30%, 10-40%, 10-50%,10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%,20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20-200%, 50-60%,50-70%, 50-80%, 50-90%, 50-100%, or 50-200%, relative to activity of thepromoter in the absence of the tumor microenvironment.

In some embodiments, the activity of the promoter is increased ordecreased by at least 2 fold (e.g., 2, 3, 4, 5, 10, 25, 20, 25, 50, or100 fold), relative to activity of the promoter in the absence of thetumor microenvironment. For example, the activity of the promoter isincreased or decreased by at least 3 fold, at least 5 fold, at least 10fold, at least 20 fold, at least 50 fold, or at least 100 fold, relativeto activity of the promoter in the absence of the tumormicroenvironment. In some embodiments, the activity of the promoter isincreased or decreased by 2-10, 2-20, 2-30, 2-40, 2-50, 2-60, 2-70,2-80, 2-90, or 2-100 fold, relative to activity of the promoter in theabsence of the tumor microenvironment.

In some embodiments, a promoter of the present disclosure is activatedunder a hypoxic condition. A “hypoxic condition” is a condition wherethe body or a region of the body is deprived of adequate oxygen supplyat the tissue level. Hypoxic conditions can cause inflammation (e.g.,the level of inflammatory cytokines increase under hypoxic conditions).In some embodiments, the promoter that is activated under hypoxiccondition is operably linked to a nucleotide encoding an effectormolecule that decreases the expression of activity of inflammatorycytokines, thus reducing the inflammation caused by the hypoxiccondition. In some embodiments, the promoter that is activated underhypoxic conditions comprises a hypoxia responsive element (HRE). A“hypoxia responsive element (HRE)” is a response element that respondsto hypoxia-inducible factor (HIF). The HRE, in some embodiments,comprises a consensus motif NCGTG (where N is either A or G).

In some embodiments, engineered cells produce multiple effectormolecules. For example, cells may be engineered to produce 2-20different effector molecules. In some embodiments, Cells engineered toproduce 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11,2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16,3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20,4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8,4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12,5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15,6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17,7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18,8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18,9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18,10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18,11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17,12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15,13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18,15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20,18-19, or 19-20 effector molecules. In some embodiments, cells areengineered to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 effector molecules.

In some embodiments, exogenous sequences can be multicistronic, i.e.,more than one separate polypeptide (e.g., multiple effector molecules)can be produced from a single mRNA transcript. Exogenous sequences canbe multicistronic through the use of various linkers, e.g., apolynucleotide sequence encoding a first effector molecule can be linkedto a nucleotide sequence encoding a second effector molecule, such as ina first gene:linker:second gene 5′ to 3′ orientation. A linker canencode a 2A ribosome skipping element, such as T2A. Other 2A ribosomeskipping elements include, but are not limited to, E2A, P2A, and F2A. 2Aribosome skipping elements allow production of separate polypeptidesencoded by the first and second genes are produced during translation. Alinker can encode a cleavable linker polypeptide sequence, such as aFurin cleavage site or a TEV cleavage site, wherein following expressionthe cleavable linker polypeptide is cleaved such that separatepolypeptides encoded by the first and second genes are produced. Acleavable linker can include a polypeptide sequence, such as such aflexible linker (e.g., a Gly-Ser-Gly sequence), that further promotescleavage.

A linker can encode an Internal Ribosome Entry Site (IRES), such thatseparate polypeptides encoded by the first and second genes are producedduring translation. A linker can encode a splice acceptor, such as aviral splice acceptor.

A linker can be a combination of linkers, such as a Furin-2A linker thatcan produce separate polypeptides through 2A ribosome skipping followedby further cleavage of the Furin site to allow for complete removal of2A residues. In some embodiments, a combination of linkers can include aFurin sequence, a flexible linker, and 2A linker. Accordingly, in someembodiments, the linker is a Furin-Gly-Ser-Gly-2A fusion polypeptide. Insome embodiments, a linker of the present disclosure is aFurin-Gly-Ser-Gly-T2A fusion polypeptide.

In general, a multicistronic system can use any number or combination oflinkers, to express any number of genes or portions thereof (e.g., anexogenous sequence can encode a first, a second, and a third effectormolecule, each separated by linkers such that separate polypeptidesencoded by the first, second, and third effector molecules areproduced).

Exogenous sequences can use multiple promoters to express genes frommultiple ORFs, i.e., more than one separate mRNA transcript can beproduced from the exogenous sequence. For example, a first promoter canbe operably linked to a polynucleotide sequence encoding a firsteffector molecule, and a second promoter can be operably linked to apolynucleotide sequence encoding a second effector molecule.

“Linkers,” as used herein can refer to polypeptides that link a firstpolypeptide sequence and a second polypeptide sequence, themulticistronic linkers described above, or the additional promoters thatare operably linked to additional ORFs described above.

Engineered cells, such as MSCs, of the present disclosure typicallyproduce multiple effector molecules, at least two of which modulatedifferent tumor-mediated immunosuppressive mechanisms. In someembodiments, at least one of the effector molecules stimulates aninflammatory pathway in the tumor microenvironment, and at least one ofthe effector molecules inhibits a negative regulator of inflammation inthe tumor microenvironment.

A “tumor microenvironment” is the cellular environment in which a tumorexists, including surrounding blood vessels, immune cells, fibroblasts,bone marrow-derived inflammatory cells, lymphocytes, signaling moleculesand the extracellular matrix (ECM) (see, e.g., Pattabiraman, D. R. &Weinberg, R. A. Nature Reviews Drug Discovery 13, 497-512 (2014);Balkwill, F. R. et al. J Cell Sci 125, 5591-5596, 2012; and Li, H. etal. J Cell Biochem 101(4), 805-15, 2007).

In some embodiments, cells are engineered to produce at least one homingmolecule. “Homing,” refers to active navigation (migration) of a cell toa target site (e.g., a cell, tissue (e.g., tumor), or organ). A “homingmolecule” refers to a molecule that directs cells to a target site. Insome embodiments, a homing molecule functions to recognize and/orinitiate interaction of a cell to a target site. Non-limiting examplesof homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2,CCR4, FPR2, VEGFR, IL6R, CXCR1, CSCR7, and PDGFR.

In some embodiments, a homing molecule is a chemokine receptor (cellsurface molecule that binds to a chemokine). Chemokines are smallcytokines or signaling proteins secreted by cells that can inducedirected chemotaxis in cells. Chemokines can be classified into fourmain subfamilies: CXC, CC, CX3C and XC, all of which exert biologicaleffects by binding selectively to chemokine receptors located on thesurface of target cells. In some embodiments, cells are engineered toproduce CXCR4, a chemokine receptor which allows cells to home along achemokine gradient towards a stromal cell-derived factor 1 (also knownas SDF1, C-X-C motif chemokine 12, and CXCL12)—expressing cell, tissue,or tumor. Non-limiting examples of chemokine receptors that may beproduced by the engineered cells of the present disclosure include: CXCchemokine receptors (e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, andCXCR7), CC chemokine receptors (CCR1, CCR2, CCR3, CCR4, CCR5, CCR6,CCR7, CCR8, CCR9, CCR10, and CCR11), CX3C chemokine receptors (e.g.,CX3CR1, which binds to CX3CL1), and XC chemokine receptors (e.g., XCR1).In some embodiments, a chemokine receptor is a G protein-linkedtransmembrane receptor, or a member of the tumor necrosis factor (TNF)receptor superfamily (including but not limited to TNFRSF1A, TNFRSF1B).In some embodiments, cells are engineered to produce CXCL8, CXCL9,and/or CXCL10, 11 or a fusion protein that encompass CXCL10 and CXCL11(promote T-cell recruitment), CCL3 and/or CXCL5, CCL21 (Th1 recruitmentand polarization). In some embodiments cells are engineered to produceCXCL13 to promote B-cell recruitment.

In some embodiments, cells are engineered to produce G-protein coupledreceptors (GPCRs) that detect N-formylated-containing oligopeptides(including but not limited to FPR2 and FPRL1).

In some embodiments, cells are engineered to produce receptors thatdetect interleukins (including but not limited to IL6R).

In some embodiments, cells are engineered to produce receptors thatdetect growth factors secreted from other cells, tissues, or tumors(including but not limited to FGFR, PDGFR, EGFR, and receptors of theVEGF family, including but not limited to VEGF-C and VEGF-D).

In some embodiments, a homing molecule is an integrin. Integrins aretransmembrane receptors that facilitate cell-extracellular matrix (ECM)adhesion. Integrins are obligate heterodimers having two subunits: α(alpha) and β (beta). The a subunit of an integrin may be, withoutlimitation: ITGA1, ITGA2, ITGA3, ITGA4, ITGA5, ITGA6, IGTA7, ITGA8,ITGA9, IGTA10, IGTA11, ITGAD, ITGAE, ITGAL, ITGAM, ITGAV, ITGA2B, ITGAX.The 13 subunit of an integrin may be, without limitation: ITGB1, ITGB2,ITGB3, ITGB4, ITGB5, ITGB6, ITGB7, and ITGB8. Cells of the presentdisclosure may be engineered to produce any combination of the integrinα and β subunits.

In some embodiments, a homing molecule is a matrix metalloproteinase(MMP). MMPs are enzymes that cleave components of the basement membraneunderlying the endothelial cell wall. Non-limiting examples of MMPsinclude MMP-2, MMP-9, and MMP. In some embodiments, cells are engineeredto produce an inhibitor of a molecule (e.g., protein) that inhibitsMMPs. For example, cells may be engineered to express an inhibitor(e.g., an RNAi molecule) of membrane type 1 MMP (MT1-MMP) or TIMPmetallopeptidase inhibitor 1 (TIMP-1).

In some embodiments, a homing molecule is a ligand that binds toselectin (e.g., hematopoietic cell E-/L-selectin ligand (HCELL), Dykstraet al., Stem Cells. 2016 October; 34(10):2501-2511) on the endotheliumof a target tissue, for example.

The term “homing molecule” also encompasses transcription factors thatregulate the production of molecules that improve/enhance homing ofcells.

In some embodiments, cell homing is increased by locally irradiating atumor/cancer cells in a subject. Radiological tissue damage aids in cellhoming, as well as endogenous T cell homing to that damaged tissue.

Examples of Engineered Cells

Cells (e.g., MSCs) as provided herein are engineered to produce multipleeffector molecules, at least two of which modulate differenttumor-mediated immunosuppressive mechanisms. In some embodiments, atleast one (e.g., 1, 2, 3, 4, 5, or more) effector molecule stimulates atleast one immunostimulatory mechanism in the tumor microenvironment, orinhibits at least one immunosuppressive mechanism in the tumormicroenvironment. In some embodiments, at least one (e.g., 1, 2, 3, 4,5, or more) effector molecule inhibits at least one immunosuppressivemechanism in the tumor microenvironment, and at least one effectormolecule (e.g., 1, 2, 3, 4, 5, or more) inhibits at least oneimmunosuppressive mechanism in the tumor microenvironment. In yet otherembodiments, at least two (e.g., 2, 3, 4, 5, or more) effector moleculesstimulate at least one immunostimulatory mechanism in the tumormicroenvironment. In still other embodiments, at least two (e.g., 1, 2,3, 4, 5, or more) effector molecules inhibit at least oneimmunosuppressive mechanism in the tumor microenvironment.

In some embodiments, a cell (e.g., MSC) is engineered to produce atleast one effector molecule that stimulates T cell signaling, activityand/or recruitment. In some embodiments, a cell (e.g., MSC) isengineered to produce at least one effector molecule that stimulatesantigen presentation and/or processing. In some embodiments, a cell(e.g., MSC) is engineered to produce at least one effector molecule thatstimulates natural killer cell-mediated cytotoxic signaling, activityand/or recruitment. In some embodiments, a cell (e.g., MSC) isengineered to produce at least one effector molecule that stimulatesdendritic cell differentiation and/or maturation. In some embodiments, acell (e.g., MSC) is engineered to produce at least one effector moleculethat stimulates immune cell recruitment. In some embodiments, a cell(e.g., MSC) is engineered to produce at least one effector molecule thatstimulates M1 macrophage signaling, activity and/or recruitment. In someembodiments, a cell (e.g., MSC) is engineered to produce at least oneeffector molecule that stimulates Th1 polarization. In some embodiments,a cell (e.g., MSC) is engineered to produce at least one effectormolecule that stimulates stroma degradation. In some embodiments, a cell(e.g., MSC) is engineered to produce at least one effector molecule thatstimulates immunostimulatory metabolite production. In some embodiments,a cell (e.g., MSC) is engineered to produce at least one effectormolecule that stimulates Type I interferon signaling. In someembodiments, a cell (e.g., MSC) is engineered to produce at least oneeffector molecule that inhibits negative costimulatory signaling. Insome embodiments, a cell (e.g., MSC) is engineered to produce at leastone effector molecule that inhibits pro-apoptotic signaling (e.g., viaTRAIL) of anti-tumor immune cells. In some embodiments, a cell (e.g.,MSC) is engineered to produce at least one effector molecule thatinhibits T regulatory (T_(reg)) cell signaling, activity and/orrecruitment. In some embodiments, a cell (e.g., MSC) is engineered toproduce at least one effector molecule that inhibits tumor checkpointmolecules. In some embodiments, a cell (e.g., MSC) is engineered toproduce at least one effector molecule that activates stimulator ofinterferon genes (STING) signaling. In some embodiments, a cell (e.g.,MSC) is engineered to produce at least one effector molecule thatinhibits myeloid-derived suppressor cell signaling, activity and/orrecruitment. In some embodiments, a cell (e.g., MSC) is engineered toproduce at least one effector molecule that degrades immunosuppressivefactors/metabolites. In some embodiments, a cell (e.g., MSC) isengineered to produce at least one effector molecule that inhibitsvascular endothelial growth factor signaling. In some embodiments, acell (e.g., MSC) is engineered to produce at least one effector moleculethat directly kills tumor cells (e.g., granzyme, perforin, oncolyticviruses, cytolytic peptides and enzymes, anti-tumor antibodies, e.g.,that trigger ADCC).

In some embodiments, at least one effector molecule: stimulates T cellsignaling, activity and/or recruitment, stimulates antigen presentationand/or processing, stimulates natural killer cell-mediated cytotoxicsignaling, activity and/or recruitment, stimulates dendritic celldifferentiation and/or maturation, stimulates immune cell recruitment,stimulates macrophage signaling, stimulates stroma degradation,stimulates immunostimulatory metabolite production, or stimulates Type Iinterferon signaling; and at least one effector molecule inhibitsnegative costimulatory signaling, inhibits pro-apoptotic signaling ofanti-tumor immune cells, inhibits T regulatory (Treg) cell signaling,activity and/or recruitment, inhibits tumor checkpoint molecules,activates stimulator of interferon genes (STING) signaling, inhibitsmyeloid-derived suppressor cell signaling, activity and/or recruitment,degrades immunosuppressive factors/metabolites, inhibits vascularendothelial growth factor signaling, or directly kills tumor cells.

In some embodiments, a cell (e.g., MSC) is engineered to produce atleast one effector molecule selected from IL-12, IFN-β, IFN-γ, IL-2,IL-15, IL-7, IL-36γ, IL-18, IL-1β, OX40-ligand, and CD40L; and/or atleast one effector molecule selected from anti-PD-1 antibodies,anti-PD-L1 antibodies, anti-CTLA-4 antibodies, and anti-IL-35antibodies; and/or at least one effector molecule selected from MIP1α(CCL3), MIP1β (CCLS), and CCL21; and/or at least one effector moleculeselected from CpG oligodeoxynucleotides; and/or at least one effectormolecule selected from microbial peptides.

In some embodiments, a cell (e.g., MSC) is engineered to produce IFN-βand at least one effector molecule selected from cytokines, antibodies,chemokines, nucleotides, peptides, enzymes, and stimulators ofinterferon genes (STINGs). In some embodiments, a cell (e.g., MSC) isengineered to produce IFN-β and at least one cytokine or receptor/ligand(e.g., IL-12, IFN-γ, IL-2, IL-15, IL-7, IL-36γ, IL-18, IL-1β,OX40-ligand, and/or CD40L).

In some embodiments, a cell (e.g., MSC) is engineered to produce IFN-βand at least one cytokine or receptor/ligand (e.g., IL-12, IFN-γ, IL-2,IL-15, IL-7, IL-36γ, IL-18, IL-1β, OX40-ligand, and/or CD40L).

In some embodiments the cytokine is produced as an engineered fusionprotein with an antibody, antibody-fragment, or receptor that self-bindsto the cytokine to induce cell-specific targeted binding such as withIL-2 fused to an antibody fragment preventing it from binding to Tregcells and preferentially binding to CD8 and NK cells. In someembodiments, a cell (e.g., MSC) is engineered to produce IFN-β and atleast one antibody (e.g., anti-PD-1 antibody, anti-PD-L1 antibody,anti-CTLA-4 antibody, anti-VEGF, anti-TGF-β, anti-IL-10, anti-TNF-α,and/or anti-IL-35 antibody). In some embodiments, a cell (e.g., MSC) isengineered to produce IFN-β and at least one chemokine (MIP1α (CCL3),MIP1β (CCLS), and/or CCL21). In some embodiments, a cell (e.g., MSC) isengineered to produce IFN-β and at least one nucleotide (e.g., a CpGoligodeoxynucleotide). In some embodiments, a cell (e.g., MSC) isengineered to produce IFN-β and at least one peptide (e.g., ananti-tumor peptide). In some embodiments, a cell (e.g., MSC) isengineered to produce IFN-β and at least one enzyme. In someembodiments, a cell (e.g., MSC) is engineered to produce IFN-β and atleast one STING activator. In some embodiments, a cell (e.g., MSC) isengineered to produce IFN-β and at least one effector with directanti-tumor activity (e.g., oncolytic virus).

In some embodiments, a cell (e.g., MSC) is engineered to produce IFN-αand MIP1-α. In some embodiments, a cell (e.g., MSC) is engineered toproduce IFN-α and MIP1-β. In some embodiments, a cell (e.g., MSC) isengineered to produce IFN-α and CXCL9. In some embodiments, a cell(e.g., MSC) is engineered to produce IFN-α and CXCL10. In someembodiments, a cell (e.g., MSC) is engineered to produce IFN-α andCXCL11. In some embodiments, a cell (e.g., MSC) is engineered to produceIFN-α and CCL21. In some embodiments, the cell is engineered to furtherproduce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL36-γ, IL-18, CD40L and/or41BB-L. In some embodiments, the cell is engineered to further produceanti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and/orOX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produce IFN-βand MIP1-α. In some embodiments, a cell (e.g., MSC) is engineered toproduce IFN-β and MIP1-β. In some embodiments, a cell (e.g., MSC) isengineered to produce IFN-β and CXCL9. In some embodiments, a cell(e.g., MSC) is engineered to produce IFN-β and CXCL10. In someembodiments, a cell (e.g., MSC) is engineered to produce IFN-β andCXCL11. In some embodiments, a cell (e.g., MSC) is engineered to produceIFN-β and CCL21. In some embodiments, the cell is engineered to furtherproduce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL36-γ, IL-18, CD40L and/or41BB-L. In some embodiments, the cell is engineered to further produceanti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and/orOX40L. In some embodiments, a cell (e.g., MSC) is engineered to produceIL-12 and MIP1-α.

In some embodiments, a cell (e.g., MSC) is engineered to produce IL-12and MIP1-β. In some embodiments, a cell (e.g., MSC) is engineered toproduce IL-12 and CXCL9. In some embodiments, a cell (e.g., MSC) isengineered to produce IL-12 and CXCL10. In some embodiments, a cell(e.g., MSC) is engineered to produce IL-12 and CXCL11. In someembodiments, a cell (e.g., MSC) is engineered to produce IL-12 andCCL21. In some embodiments, the cell is engineered to further produceIFN-β, IFN-γ, IL-2, IL-7, IL-15, IL36-γ, IL-18, CD40L and/or 41BB-L. Insome embodiments, the cell is engineered to further produce anti-CD40antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produceTNF-related apoptosis-inducing ligand (TRAIL) and MIP1-α. In someembodiments, a cell (e.g., MSC) is engineered to produce TRAIL andMIP1-β. In some embodiments, a cell (e.g., MSC) is engineered to produceTRAIL and CXCL9. In some embodiments, a cell (e.g., MSC) is engineeredto produce TRAIL and CXCL10. In some embodiments, a cell (e.g., MSC) isengineered to produce TRAIL and CXCL11. In some embodiments, a cell(e.g., MSC) is engineered to produce TRAIL and CCL21. In someembodiments, the cell is engineered to further produce IL-12, IFN-γ,IL-2, IL-7, IL-15, IL36-γ, IL-18, CD40L and/or 41BB-L. In someembodiments, the cell is engineered to further produce anti-CD40antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produce astimulator of interferon gene (STING) and MIP1-α. In some embodiments, acell (e.g., MSC) is engineered to produce STING and MIP1-β. In someembodiments, a cell (e.g., MSC) is engineered to produce STING andCXCL9. In some embodiments, a cell (e.g., MSC) is engineered to produceSTING and CXCL10. In some embodiments, a cell (e.g., MSC) is engineeredto produce STING and CXCL11. In some embodiments, a cell (e.g., MSC) isengineered to produce STING and CCL21. In some embodiments, the cell isengineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL36-γ,IL-18, CD40L and/or 41BB-L. In some embodiments, the cell is engineeredto further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produce CD40Land MIP1-α. In some embodiments, a cell (e.g., MSC) is engineered toproduce CD40L and MIP1-β. In some embodiments, a cell (e.g., MSC) isengineered to produce CD40L and CXCL9. In some embodiments, a cell(e.g., MSC) is engineered to produce CD40L and CXCL10. In someembodiments, a cell (e.g., MSC) is engineered to produce CD40L andCXCL11. In some embodiments, a cell (e.g., MSC) is engineered to produceCD40L and CCL21. In some embodiments, the cell is engineered to furtherproduce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL36-γ, IL-18, and/or 41BB-L.In some embodiments, the cell is engineered to further produce anti-CD40antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to producecytosine deaminase and MIP1-α. In some embodiments, a cell (e.g., MSC)is engineered to produce cytosine deaminase and MIP1-β. In someembodiments, a cell (e.g., MSC) is engineered to produce cytosinedeaminase and CXCL9. In some embodiments, a cell (e.g., MSC) isengineered to produce cytosine deaminase and CXCL10. In someembodiments, a cell (e.g., MSC) is engineered to produce cytosinedeaminase and CXCL11. In some embodiments, a cell (e.g., MSC) isengineered to produce cytosine deaminase and CCL21. In some embodiments,the cell is engineered to further produce IL-12, IFN-γ, IL-2, IL-7,IL-15, IL36-γ, IL-18, CD40L, and/or 41BB-L. In some embodiments, thecell is engineered to further produce anti-CD40 antibody, anti-CTLA4antibody, anti-PD-L1 antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produce IFN-αand IL-12. In some embodiments, a cell (e.g., MSC) is engineered toproduce IFN-α and IFN-γ. In some embodiments, a cell (e.g., MSC) isengineered to produce IFN-α and IL-2. In some embodiments, a cell (e.g.,MSC) is engineered to produce IFN-α and IL-7. In some embodiments, acell (e.g., MSC) is engineered to produce IFN-α and IL-15. In someembodiments, a cell (e.g., MSC) is engineered to produce IFN-α andIL-36γ. In some embodiments, a cell (e.g., MSC) is engineered to produceIFN-α and IL-18. In some embodiments, a cell (e.g., MSC) is engineeredto produce IFN-α and CD40L. In some embodiments, a cell (e.g., MSC) isengineered to produce IFN-α and 41BB-L. In some embodiments, the cell isengineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11,CXCL10-11 fusion, CXCL13 and/or CCL21. In some embodiments, the cell isengineered to further produce anti-CD40 antibody, anti-CTLA4 antibody,anti-PD-L1 antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produce IFN-βand IL-12. In some embodiments, a cell (e.g., MSC) is engineered toproduce IFN-β and IFN-γ. In some embodiments, a cell (e.g., MSC) isengineered to produce IFN-β and IL-2. In some embodiments, a cell (e.g.,MSC) is engineered to produce IFN-β and IL-7. In some embodiments, acell (e.g., MSC) is engineered to produce IFN-β and IL-15. In someembodiments, a cell (e.g., MSC) is engineered to produce IFN-β andIL-36γ. In some embodiments, a cell (e.g., MSC) is engineered to produceIFN-β and IL-18. In some embodiments, a cell (e.g., MSC) is engineeredto produce IFN-β and CD40L. In some embodiments, a cell (e.g., MSC) isengineered to produce IFN-β and 41BB-L. In some embodiments, the cell isengineered to further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11,and/or CCL21. In some embodiments, the cell is engineered to furtherproduce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1 antibody,and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produceTNF-related apoptosis-inducing ligand (TRAIL) and IL-12. In someembodiments, a cell (e.g., MSC) is engineered to produce TRAIL andIFN-γ. In some embodiments, a cell (e.g., MSC) is engineered to produceTRAIL and IL-2. In some embodiments, a cell (e.g., MSC) is engineered toproduce TRAIL and IL-7. In some embodiments, a cell (e.g., MSC) isengineered to produce TRAIL and IL-15. In some embodiments, a cell(e.g., MSC) is engineered to produce TRAIL and IL-36γ. In someembodiments, a cell (e.g., MSC) is engineered to produce TRAIL andIL-18. In some embodiments, a cell (e.g., MSC) is engineered to produceTRAIL and CD40L. In some embodiments, a cell (e.g., MSC) is engineeredto produce TRAIL and 41BB-L. In some embodiments, the cell is engineeredto further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CCL21.In some embodiments, the cell is engineered to further produce anti-CD40antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produce astimulator of interferon gene (STING) and IL-12. In some embodiments, acell (e.g., MSC) is engineered to produce STING and IFN-γ. In someembodiments, a cell (e.g., MSC) is engineered to produce STING and IL-2.In some embodiments, a cell (e.g., MSC) is engineered to produce STINGand IL-7. In some embodiments, a cell (e.g., MSC) is engineered toproduce STING and IL-15. In some embodiments, a cell (e.g., MSC) isengineered to produce STING and IL-36γ. In some embodiments, a cell(e.g., MSC) is engineered to produce STING and IL-18. In someembodiments, a cell (e.g., MSC) is engineered to produce STING andCD40L. In some embodiments, a cell (e.g., MSC) is engineered to produceSTING and 41BB-L. In some embodiments, the cell is engineered to furtherproduce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CCL21. In someembodiments, the cell is engineered to further produce anti-CD40antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produce CD40Land IL-12. In some embodiments, a cell (e.g., MSC) is engineered toproduce CD40L and IFN-γ. In some embodiments, a cell (e.g., MSC) isengineered to produce CD40L and IL-2. In some embodiments, a cell (e.g.,MSC) is engineered to produce CD40L and IL-7. In some embodiments, acell (e.g., MSC) is engineered to produce CD40L and IL-15. In someembodiments, a cell (e.g., MSC) is engineered to produce CD40L andIL-36γ. In some embodiments, a cell (e.g., MSC) is engineered to produceCD40L and IL-18. In some embodiments, a cell (e.g., MSC) is engineeredto produce CD40L and 41BB-L. In some embodiments, the cell is engineeredto further produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CCL21.In some embodiments, the cell is engineered to further produce anti-CD40antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to producecytosine deaminase and IL-12. In some embodiments, a cell (e.g., MSC) isengineered to produce cytosine deaminase and IFN-γ. In some embodiments,a cell (e.g., MSC) is engineered to produce cytosine deaminase and IL-2.In some embodiments, a cell (e.g., MSC) is engineered to producecytosine deaminase and IL-7. In some embodiments, a cell (e.g., MSC) isengineered to produce cytosine deaminase and IL-15. In some embodiments,a cell (e.g., MSC) is engineered to produce cytosine deaminase andIL-36γ. In some embodiments, a cell (e.g., MSC) is engineered to producecytosine deaminase and IL-18. In some embodiments, a cell (e.g., MSC) isengineered to produce cytosine deaminase and CD40L. In some embodiments,a cell (e.g., MSC) is engineered to produce cytosine deaminase and41BB-L. In some embodiments, the cell is engineered to further produceMIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CCL21. In someembodiments, the cell is engineered to further produce anti-CD40antibody, anti-CTLA4 antibody, anti-PD-L1 antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produce MIP1-αand IL-12. In some embodiments, a cell (e.g., MSC) is engineered toproduce MIP1-α and MIP1-γ. In some embodiments, a cell (e.g., MSC) isengineered to produce MIP1-α and IL-2. In some embodiments, a cell(e.g., MSC) is engineered to produce MIP1-α and IL-7. In someembodiments, a cell (e.g., MSC) is engineered to produce MIP1-α andIL-15. In some embodiments, a cell (e.g., MSC) is engineered to produceMIP1-α and IL-36γ. In some embodiments, a cell (e.g., MSC) is engineeredto produce MIP1-α and IL-18. In some embodiments, a cell (e.g., MSC) isengineered to produce MIP1-α and CD40L. In some embodiments, a cell(e.g., MSC) is engineered to produce MIP1-α and 41BB-L. In someembodiments, the cell is engineered to further produce IFN-α, IFN-β,TRAIL, STING, CD40L, and/or cytosine deaminase. In some embodiments, thecell is engineered to further produce anti-CD40 antibody, anti-CTLA4antibody, anti-PD-L1 antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produce MIP1-βand IL-12. In some embodiments, a cell (e.g., MSC) is engineered toproduce MIP1-β and MIP1-γ. In some embodiments, a cell (e.g., MSC) isengineered to produce MIP1-β and IL-2. In some embodiments, a cell(e.g., MSC) is engineered to produce MIP1-β and IL-7. In someembodiments, a cell (e.g., MSC) is engineered to produce MIP1-β andIL-15. In some embodiments, a cell (e.g., MSC) is engineered to produceMIP1-β and IL-36γ. In some embodiments, a cell (e.g., MSC) is engineeredto produce MIP1-β and IL-18. In some embodiments, a cell (e.g., MSC) isengineered to produce MIP1-β and CD40L. In some embodiments, a cell(e.g., MSC) is engineered to produce MIP1-β and 41BB-L. In someembodiments, the cell is engineered to further produce IFN-α, IFN-β,TRAIL, STING, CD40L, and/or cytosine deaminase. In some embodiments, thecell is engineered to further produce anti-CD40 antibody, anti-CTLA4antibody, anti-PD-L1 antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produce CXCL9and IL-12. In some embodiments, a cell (e.g., MSC) is engineered toproduce CXCL9 and IFN-γ. In some embodiments, a cell (e.g., MSC) isengineered to produce CXCL9 and IL-2. In some embodiments, a cell (e.g.,MSC) is engineered to produce CXCL9 and IL-7. In some embodiments, acell (e.g., MSC) is engineered to produce CXCL9 and IL-15. In someembodiments, a cell (e.g., MSC) is engineered to produce CXCL9 andIL-36γ. In some embodiments, a cell (e.g., MSC) is engineered to produceCXCL9 and IL-18. In some embodiments, a cell (e.g., MSC) is engineeredto produce CXCL9 and CD40L. In some embodiments, a cell (e.g., MSC) isengineered to produce CXCL9 and 41BB-L. In some embodiments, the cell isengineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/orcytosine deaminase. In some embodiments, the cell is engineered tofurther produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produce aCXCL10 and IL-12. In some embodiments, a cell (e.g., MSC) is engineeredto produce CXCL10 and IFN-γ. In some embodiments, a cell (e.g., MSC) isengineered to produce CXCL10 and IL-2. In some embodiments, a cell(e.g., MSC) is engineered to produce CXCL10 and IL-7. In someembodiments, a cell (e.g., MSC) is engineered to produce CXCL10 andIL-15. In some embodiments, a cell (e.g., MSC) is engineered to produceCXCL10 and IL-36γ. In some embodiments, a cell (e.g., MSC) is engineeredto produce CXCL10 and IL-18. In some embodiments, a cell (e.g., MSC) isengineered to produce CXCL10 and CD40L. In some embodiments, a cell(e.g., MSC) is engineered to produce CXCL10 and 41BB-L. In someembodiments, the cell is engineered to further produce IFN-α, IFN-β,TRAIL, STING, CD40L, and/or cytosine deaminase. In some embodiments, thecell is engineered to further produce anti-CD40 antibody, anti-CTLA4antibody, anti-PD-L1 antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produce CXCL11and IL-12. In some embodiments, a cell (e.g., MSC) is engineered toproduce CXCL11 and IFN-γ. In some embodiments, a cell (e.g., MSC) isengineered to produce CXCL11 and IL-2. In some embodiments, a cell(e.g., MSC) is engineered to produce CXCL11 and IL-7. In someembodiments, a cell (e.g., MSC) is engineered to produce CXCL11 andIL-15. In some embodiments, a cell (e.g., MSC) is engineered to produceCXCL11 and IL-36γ. In some embodiments, a cell (e.g., MSC) is engineeredto produce CXCL11 and IL-18. In some embodiments, a cell (e.g., MSC) isengineered to produce CXCL11 and 41BB-L. In some embodiments, the cellis engineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L,and/or cytosine deaminase. In some embodiments, the cell is engineeredto further produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produce CCL21and IL-12. In some embodiments, a cell (e.g., MSC) is engineered toproduce CCL21 and IFN-γ. In some embodiments, a cell (e.g., MSC) isengineered to produce CCL21 and IL-2. In some embodiments, a cell (e.g.,MSC) is engineered to produce CCL21 and IL-7. In some embodiments, acell (e.g., MSC) is engineered to produce CCL21 and IL-15. In someembodiments, a cell (e.g., MSC) is engineered to produce CCL21 andIL-36γ. In some embodiments, a cell (e.g., MSC) is engineered to produceCCL21 and IL-18. In some embodiments, a cell (e.g., MSC) is engineeredto produce CCL21 and CD40L. In some embodiments, a cell (e.g., MSC) isengineered to produce CCL21 and 41BB-L. In some embodiments, the cell isengineered to further produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/orcytosine deaminase. In some embodiments, the cell is engineered tofurther produce anti-CD40 antibody, anti-CTLA4 antibody, anti-PD-L1antibody, and/or OX40L.

In some embodiments, a cell (e.g., MSC) is engineered to produce IFN-αand anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce IFN-α and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce IFN-α and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce IFN-α andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CXCL21. Insome embodiments, the cell is engineered to further produce IL-12,IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and/or 41BB-L.

In some embodiments, a cell (e.g., MSC) is engineered to produce IFN-βand anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce IFN-β and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce IFN-β and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce IFN-β andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CXCL21. Insome embodiments, the cell is engineered to further produce IL-12,IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and/or 41BB-L.

In some embodiments, a cell (e.g., MSC) is engineered to produce TRAILand anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce TRAIL and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce TRAIL and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce TRAIL andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CXCL21. Insome embodiments, the cell is engineered to further produce IL-12,IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and/or 41BB-L.

In some embodiments, a cell (e.g., MSC) is engineered to produce STINGand anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce STING and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce STING and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce STING andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CXCL21. Insome embodiments, the cell is engineered to further produce IL-12,IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and/or 41BB-L.

In some embodiments, a cell (e.g., MSC) is engineered to produce CD40Land anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce CD40L and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce CD40L and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce CD40L andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CXCL21. Insome embodiments, the cell is engineered to further produce IL-12,IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and/or 41BB-L.

In some embodiments, a cell (e.g., MSC) is engineered to producecytosine deaminase and anti-PD-L1 antibody. In some embodiments, a cell(e.g., MSC) is engineered to produce cytosine deaminase and OX40L. Insome embodiments, a cell (e.g., MSC) is engineered to produce cytosinedeaminase and anti-CTLA4 antibody. In some embodiments, a cell (e.g.,MSC) is engineered to produce cytosine deaminase and anti-CD47 antibody.In some embodiments, the cell is engineered to further produce MIP1-α,MIP1-β, CXCL9, CXCL10, CXCL11, and/or CXCL21. In some embodiments, thecell is engineered to further produce IL-12, IFN-γ, IL-2, IL-7, IL-15,IL-36γ, IL-18, CD40L, and/or 41BB-L.

In some embodiments, a cell (e.g., MSC) is engineered to produce MIP1-αand anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce MIP1-α and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce MIP1-α and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce MIP1-α andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/or cytosinedeaminase. In some embodiments, the cell is engineered to furtherproduce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and/or41BB-L.

In some embodiments, a cell (e.g., MSC) is engineered to produce MIP1-βand anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce MIP1-β and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce MIP1-β and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce MIP1-β andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/or cytosinedeaminase. In some embodiments, the cell is engineered to furtherproduce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and/or41BB-L.

In some embodiments, a cell (e.g., MSC) is engineered to produce CXCL9and anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce CXCL9 and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce CXCL9 and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce CXCL9 andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/or cytosinedeaminase. In some embodiments, the cell is engineered to furtherproduce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and/or41BB-L.

In some embodiments, a cell (e.g., MSC) is engineered to produce CXCL10and anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce CXCL10 and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce CXCL10 and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce CXCL10 andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/or cytosinedeaminase. In some embodiments, the cell is engineered to furtherproduce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and/or41BB-L.

In some embodiments, a cell (e.g., MSC) is engineered to produce CXCL11and anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce CXCL11 and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce CXCL11 and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce CXCL11 andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/or cytosinedeaminase. In some embodiments, the cell is engineered to furtherproduce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and/or41BB-L.

In some embodiments, a cell (e.g., MSC) is engineered to produce CCL21and anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce CCL21 and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce CCL21 and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce CCL21 andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/or cytosinedeaminase. In some embodiments, the cell is engineered to furtherproduce IL-12, IFN-γ, IL-2, IL-7, IL-15, IL-36γ, IL-18, CD40L, and/or41BB-L.

In some embodiments, a cell (e.g., MSC) is engineered to produce IL-12and anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce IL-12 and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce IL-12 and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce IL-12 andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/or cytosinedeaminase. In some embodiments, the cell is engineered to furtherproduce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CCL21.

In some embodiments, a cell (e.g., MSC) is engineered to produce IFN-γand anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce IFN-γ and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce IFN-γ and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce IFN-γ andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/or cytosinedeaminase. In some embodiments, the cell is engineered to furtherproduce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CCL21.

In some embodiments, a cell (e.g., MSC) is engineered to produce IL-2and anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce IL-2 and OX40L. In some embodiments, a cell (e.g.,MSC) is engineered to produce IL-2 and anti-CTLA4 antibody. In someembodiments, a cell (e.g., MSC) is engineered to produce IL-2 andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/or cytosinedeaminase. In some embodiments, the cell is engineered to furtherproduce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CCL21.

In some embodiments, a cell (e.g., MSC) is engineered to produce IL-7and anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce IL-7 and OX40L. In some embodiments, a cell (e.g.,MSC) is engineered to produce IL-7 and anti-CTLA4 antibody. In someembodiments, a cell (e.g., MSC) is engineered to produce IL-7 andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/or cytosinedeaminase. In some embodiments, the cell is engineered to furtherproduce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CCL21.

In some embodiments, a cell (e.g., MSC) is engineered to produce IL-15and anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce IL-15 and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce IL-15 and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce IL-15 andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/or cytosinedeaminase. In some embodiments, the cell is engineered to furtherproduce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CCL21.

In some embodiments, a cell (e.g., MSC) is engineered to produce IL-36-γand anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce IL-36-γ and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce IL-36-γ and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce IL-36-γand anti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/or cytosinedeaminase. In some embodiments, the cell is engineered to furtherproduce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CCL21.

In some embodiments, a cell (e.g., MSC) is engineered to produce IL-18and anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce IL-18 and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce IL-18 and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce IL-18 andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/or cytosinedeaminase. In some embodiments, the cell is engineered to furtherproduce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CCL21.

In some embodiments, a cell (e.g., MSC) is engineered to produce CD40Land anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce CD40L and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce CD40L and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce CD40L andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/or cytosinedeaminase. In some embodiments, the cell is engineered to furtherproduce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CCL21.

In some embodiments, a cell (e.g., MSC) is engineered to produce 41BB-Land anti-PD-L1 antibody. In some embodiments, a cell (e.g., MSC) isengineered to produce 41BB-L and OX40L. In some embodiments, a cell(e.g., MSC) is engineered to produce 41BB-L and anti-CTLA4 antibody. Insome embodiments, a cell (e.g., MSC) is engineered to produce 41BB-L andanti-CD47 antibody. In some embodiments, the cell is engineered tofurther produce IFN-α, IFN-β, TRAIL, STING, CD40L, and/or cytosinedeaminase. In some embodiments, the cell is engineered to furtherproduce MIP1-α, MIP1-β, CXCL9, CXCL10, CXCL11, and/or CCL21.

Secretion Signals

In general, the one or more effector molecules comprise a secretionsignal peptide (also referred to as a signal peptide or signal sequence)at the effector molecule's N-terminus that direct newly synthesizedproteins destined for secretion or membrane insertion to the properprotein processing pathways. The secretion signal peptide operablyassociated with a effector molecule can be a native secretion signalpeptide native secretion signal peptide (e.g., the secretion signalpeptide generally endogenously associated with the given effectormolecule). The secretion signal peptide operably associated with aeffector molecule can be a non-native secretion signal peptide nativesecretion signal peptide. Non-native secretion signal peptides canpromote improved expression and function, such as maintained secretion,in particular environments, such as tumor microenvironments.Non-limiting examples of non-native secretion signal peptide are shownin Table 5.

TABLE 5 Exemplary Signal Secretion Peptides Protein Source Name SEQUENCE(Uniprot) DNA SEQUENCE IL-12 MCHQQLV P29460 ATGTGTCACCAGC ISWFSLVAGCTCGTTATAT FLASPLVA CCTGGTTTAGTTT (SEQ ID GGTGTTTCTCGCT NO: 112)TCACCCCTGGTGG CA (SEQ ID NO: 31) IL-12 MCHQQLV — ATGTGCCATCAG (CodonISWFSL CAACTCGTCATCT Optimized) VFLASPL CCTGGTTCTCCCT VA TGTGTTCCTCGCT(SEQ ID TCCCCTCTGGTCG NO: 112) CC (SEQ ID NO: 32) IL-2 MQLLSCI —ATGCAACTGCTGT (Optimized) ALILALV CATGTATCGCAC (SEQ ID TCATCCTGGCGCTNO: 113) GGTA (SEQ ID NO: 33) IL-2 MYRMQLL P60568 ATGTATCGGATGCA(Native) SCIALS ACTTTTGAGCTGCA LALVTNS TCGCATTGTCTCTG (SEQ ID GCGCTGGTNO: 114) GACAAATTCC (SEQ ID NO: 34) Trypsinogen-2 MNLLLIL P07478ATGAATCTCTTGC TFVAAA TCATACTTACGT VA TTGTCGCTGCTGC (SEQ ID CGTTGCGNO: 115) (SEQ ID NO: 35) Gaussia MGVKVLF — ATGGGCGTGAAGG LuciferaseALICIA TCTTGTTTGCCC VAEA TTATCTGCATAGC (SEQ ID TGTTGCGGAGGC NO: 116) G(SEQ ID NO: 36) CD5 MPMGSL P06127 ATGCCGATGGG QPLATLY GAGCCTTCAACLLGMLVA CTTTGGCAACG SCLG CTTTATCTTCT (SEQ ID GGGGATGTTGG NO: 117)TTGCTAGTTGC CTTGGG (SEQ ID NO: 37) IgKVII METDTLL ATGGAAACTGA (mouse)LWVLLL CACGTTGTTGC WVPGSTGD TGTGGGTATTG (SEQ ID CTCTTGTGGGT NO: 118)CCCAGGATCTA CGGGCGAC (SEQ ID NO: 38) IgKVII MDMRVPA P01597 ATGGATATGAGG(human) QLLGL GTTCCCGCCCAG LLLWLRG CTTTTGGGGCTG ARC CTTTTGTTGTGG (SEQ IDCTTCGAGGGGCT NO: 119) CGGTGT (SEQ ID NO: 39) VSV-G MKCLLYL —ATGAAGTGTCTG AFLFIG TTGTACCTGGCG VNC TTTCTGTTCATT (SEQ ID GGTGTAAACTGTNO: 120) (SEQ ID NO: 40) Prolactin MNIKGSP P01236 ATGAATATCAAA WKGSLLGGAAGTCCGTGG LLLVSNL AAGGGTAGTCTC LLCQSV CTGCTGCTCCTC AP GTATCTAACCTT(SEQ ID CTCCTTTGTCAA NO: 121) TCCGTGGCACCC (SEQ ID NO: 41) Serum MKWVTFIP02768 ATGAAATGGGTAA albumin SLLFLFS CATTCATATCACT pre- SAYSTCTCTTTCTGTTC proprotein (SEQ ID AGCTCTGCGTAT NO: 122) TCT (SEQ IDNO: 42) Azurocidin MTRLTVL 20160 ATGACAAGGCTTA Pre- ALLAGL CTGTTTTGGCTCTproprotein LASSRA CCTCGCTGGACTC (SEQ ID TTGGCTTCCTCC NO: 123) CGAGCA(SEQ ID NO: 43) Osteonectin MRAWIF P09486 ATGAGGGCTTGGA (BM40) FLLCLAGTTTTTTTTCTGCT RALA CTGCCTTGCCGGT (SEQ ID CGAGCCCTGGCG NO: 124) (SEQ IDNO: 44) CD33 MPLLLL P20138 ATGCCTCTTCTGC LPLLWAG TTTTGCTTCCTCT ALATTTGTGGGCAGGT (SEQ ID GCCCTCGCA NO: 125) (SEQ ID NO: 45) IL-6 MNSFSTSP05231 ATGAACTCTTTCT AFGPVA CAACCTCTGCGTT FSLGLLL TGGTCCGGTCGCT VLPAAFPTTCTCCCTTGGGC AP TCCTGCTTGTCTT (SEQ ID GCCAGCAGCGTTT NO: 126) CCTGCGCCA(SEQ ID NO: 46) IL-8 MTSKLAV P10145 ATGACAAGTAAA ALLAAF CTGGCGGTAGCCLISAALC TTGCTCGCGGCC (SEQ ID TTTTTGATTTCC NO: 127) GCAGCCCTTTGT (SEQ IDNO: 47) CCL2 MKVSAAL P13500 ATGAAGGTAAGT LCLLLI GCAGCGTTGCTT AATFIPTGCCTTCTCCTC QGLA ATTGCAGCGACC (SEQ ID TTTATTCCTCAA NO: 128) GGGCTGGCC(SEQ ID NO: 48) TIMP2 MGAAAR P16035 ATGGGAGCGGCAG TLRLAL CTAGAACACTTCGGLLLLA ACTTGCCCTTGGG TLLRPAD CTCTTGCTCCTTG A CAACCCTCCTTAG (SEQ IDACCTGCCGACGCA NO: 129) (SEQ ID NO: 49) VEGFB MSPLLR P49765 ATGTCACCGTTGTRLLLAAL TGCGGAGATTGCT LQLAPAQA GTTGGCCGCACTT (SEQ ID TTGCAACTGGCTCNO: 130) CTGCTCAAGCC (SEQ ID NO: 50) Osteo- MNNLLC O00300 ATGAATAACCTGCprotegerin CALVFLD TCTGTTGTGCGCT ISIKWTTQ CGTGTTCCTGGAC (SEQ IDATTTCTATAAAAT NO: 131) GGACAACGCAA (SEQ ID NO: 51) Serpin E1 MQMSPAP05121 ATGCAAATGTCTC LTCLVLG CTGCCCTTACCTG LALVFGE TCTCGTACTTGGT GSACTTGCGCTCGTAT (SEQ ID TTGGAGAGGGATC NO: 132) AGCC (SEQ ID NO: 52)GROalpha MARAALS P09341 ATGGCAAGGGCTG AAPSNP CACTCAGTGCTGC RLLRVALCCCGTCTAATCCC LLLLLV AGATTGCTTCGAG AAGRRA TTGCATTGCTTCT AG TCTGTTGCTGGTT(SEQ ID GCAGCTGGTAGGA NO: 133) GAGCAGCGG GT (SEQ ID NO: 53) CXCL12MNAKVV P48061 ATGAATGCAAAAG VVLVLV TCGTGGTCGTGCT LTALCL GGTTTTGGTTCTGSDG ACGGCGTTGTGTC (SEQ ID TTAGTGATGGG NO: 134) (SEQ ID NO: 54) IL-21MERIVIC Q9HBE4 ATGGAACGCATTG (Codon LMVIFLG TGATCTGCCTGAT Optimized)TLVHKSSS GGTCATCTTCCTG (SEQ ID GGCACCTTAGTGC NO: 135) ACAAGTCGAGCAG C(SEQ ID NO: 55)Cell Types

The present disclosure refers to mesenchymal stem cells (MSCs) (e.g.,human MSCs) engineered to produce multiple effector molecules. Anengineered cell (engineered to produce effector molecules), as providedherein, may also be selected from natural killer (NK) cells, NKT cells,innate lymphoid cells, mast cells, eosinophils, basophils, macrophages,neutrophils, and dendritic cells, T cells (e.g., CD8+ T cells, CD4+ Tcells, gamma-delta T cells, and T regulatory cells (CD4⁺, FOXP3⁺,CD25⁺)) and B cells. It should be understood, however, that anyreference to MSC engineering can also be applied to other cell types(e.g., cell types of the immune system).

In some embodiments, an engineered cell (e.g., MSC) is from (e.g.,obtained from or derived from) bone marrow. In some embodiments, anengineered mesenchymal stem cell is from (e.g., obtained from or derivedfrom) adipose tissue. In some embodiments, an engineered mesenchymalstem cell is from (e.g., obtained from or derived from) an umbilicalcord. In some embodiments, engineered mesenchymal stem cell is from apluripotent stem cell (e.g., an embryonic stem cell or an inducedpluripotent stem cell).

Thus, the present disclosure provides a T cell (e.g., CD8+ T cell, CD4+T cell, gamma-delta T cell, or T regulatory cell (CD4⁺, FOXP3⁺, CD25⁺))engineered to produce multiple effector molecules, at least two of whichmodulate different tumor-mediated immunosuppressive mechanisms. In someembodiments, a B cell is engineered to produce multiple effectormolecules, at least two of which modulate different tumor-mediatedimmunosuppressive mechanisms. In some embodiments, a NK cell isengineered to produce multiple effector molecules, at least two of whichmodulate different tumor-mediated immunosuppressive mechanisms. In someembodiments, a NKT cell is engineered to produce multiple effectormolecules, at least two of which modulate different tumor-mediatedimmunosuppressive mechanisms. In some embodiments, an innate lymphoidcell is engineered to produce multiple effector molecules, at least twoof which modulate different tumor-mediated immunosuppressive mechanisms.In some embodiments, a mast cell is engineered to produce multipleeffector molecules, at least two of which modulate differenttumor-mediated immunosuppressive mechanisms. In some embodiments, aneosinophil is engineered to produce multiple effector molecules, atleast two of which modulate different tumor-mediated immunosuppressivemechanisms. In some embodiments, a basophil is engineered to producemultiple effector molecules, at least two of which modulate differenttumor-mediated immunosuppressive mechanisms. In some embodiments, amacrophage is engineered to produce multiple effector molecules, atleast two of which modulate different tumor-mediated immunosuppressivemechanisms. In some embodiments, a neutrophil is engineered to producemultiple effector molecules, at least two of which modulate differenttumor-mediated immunosuppressive mechanisms. In some embodiments, adendritic cell is engineered to produce multiple effector molecules, atleast two of which modulate different tumor-mediated immunosuppressivemechanisms.

In some embodiments, at least one of the effector molecules stimulatesan immunostimulatory mechanism in the tumor microenvironment and/orinhibits an immunosuppressive mechanism in the tumor microenvironment.

In some embodiments, at least one of the effector molecules (a)stimulates T cell signaling, activity and/or recruitment, (b) stimulatesantigen presentation and/or processing, (c) stimulates natural killercell-mediated cytotoxic signaling, activity and/or recruitment, (d)stimulates dendritic cell differentiation and/or maturation, (e)stimulates immune cell recruitment, (f) stimulates pro-inflammatorymacrophage signaling, activity and/or recruitment or inhibitsanti-inflammatory macrophage signaling, activity and/or recruitment, (g)stimulates stroma degradation, (h) stimulates immunostimulatorymetabolite production, (i) stimulates Type I interferon signaling, (j)inhibits negative costimulatory signaling, (k) inhibits pro-apoptoticsignaling of anti-tumor immune cells, (l) inhibits T regulatory(T_(reg)) cell signaling, activity and/or recruitment, (m) inhibitstumor checkpoint molecules, (n) stimulates stimulator of interferongenes (STING) signaling, (o) inhibits myeloid-derived suppressor cellsignaling, activity and/or recruitment, (p) degrades immunosuppressivefactors/metabolites, (q) inhibits vascular endothelial growth factorsignaling, and/or (r) directly kills tumor cells.

Methods

Also provided herein are methods that include culturing the engineeredMSCs (or other engineered immune cell) of the present disclosure.Methods of culturing MSCs are known. In some embodiments, MSCs areculture in growth medium (e.g., MSCGM human Mesenchymal Stem Cell GrowthBULLETKIT™ Medium (serum containing), THERAPEAK™ MSCGM-CD™ MesenchymalStem Cell Chemically Defined Medium (serum free), or RoosterBioxeno-free MSC media). Methods of culturing other cells, such as immunecells, are known to those skilled in the art.

Further provided herein are methods that include delivering, oradministering, to a subject (e.g., a human subject) engineered cells asprovided herein to produce in vivo at least one effector moleculeproduced by the cells. In some embodiments, the cells are administeredvia intravenous, intraperitoneal, intratracheal, subcutaneous,intratumoral, oral, anal, intranasal (e.g., packed in a deliveryparticle), or arterial (e.g., internal carotid artery) routes. Thus, thecells may be administered systemically or locally (e.g., to a TME). Theengineered cells or polynucleotides described herein can be in acomposition containing a pharmaceutically acceptable carrier, e.g., anaqueous carrier. A variety of aqueous carriers can be used, e.g., water,buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like.These compositions can be sterilized by conventional, well knownsterilization techniques, or can be sterile filtered. The resultingaqueous solutions can be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

Some methods comprise selecting a subject (or patient population) havinga tumor (or cancer) and treating that subject with engineered cells.

The engineered cells of the present disclosure may be used, in someinstances, to treat cancer, such as ovarian cancer. Other cancers aredescribed herein. For example, the engineered cells may be used to treatbladder tumors, brain tumors, breast tumors, cervical tumors, colorectaltumors, esophageal tumors, gliomas, kidney tumors, liver tumors, lungtumors, melanomas, ovarian tumors, pancreatic tumors, prostate tumors,skin tumors, thyroid tumors, and/or uterine tumors.

The methods provided herein also include delivering a preparation ofengineered cells, such as engineered cells. A preparation, in someembodiments, is a substantially pure preparation, containing, forexample, less than 5% (e.g., less than 4%, 3%, 2%, or 1%) of cells otherthan cells. A preparation may comprise 1×10⁵ cells/kg to 1×10⁷ cells/kg,such as engineered cells.

The methods provided herein also include delivering a composition invivo capable of producing the engineered cells described herein, such asdelivering a lentivirus in vivo. Other in vivo delivery mechanisms andsystems can also be used, including those known for use in humantherapy, such as viral delivery systems (e.g., retroviral or adenoviralsystems), transposons (e.g., Sleeping Beauty and PiggyBac transposonsystems), integrated using PhiC31 into genomic pseudosites, or usingnucleases, such as zinc fingers (ZFs), clustered regularly interspacedshort palindromic repeats (CRISPR), or transcription activator-likeeffector nucleases (TALENs).

TABLE 6 Sequences encoding exemplary effector moleculesIL12 (Human) (SEQ ID NO: 56)ATGTGCCATCAGCAGCTTGTCATATCTTGGTTTTCACTTGTATTCCTGGCCAGCCCTTTGGTTGCGATCTGGGAGCTCAAGAAGGATGTGTACGTTGTAGAGCTGGACTGGTACCCCGATGCTCCCGGTGAGATGGTCGTTTTGACATGTGACACTCCAGAAGAGGACGGTATTACGTGGACTCTGGACCAGTCCTCCGAAGTTCTTGGTTCTGGTAAGACTCTGACTATCCAGGTGAAAGAATTTGGGGATGCGGGACAATACACATGCCACAAGGGAGGCGAGGTGTTGTCTCATAGTTTGCTGCTTCTCCACAAGAAAGAGGATGGAATCTGGAGCACCGACATACTCAAGGATCAAAAGGAACCCAAAAATAAGACATTTCTGCGATGTGAGGCTAAGAACTATAGTGGCCGCTTCACTTGTTGGTGGCTGACTACCATCAGCACAGATCTCACGTTTTCAGTAAAAAGTAGTAGAGGTTCAAGTGATCCTCAAGGGGTAACGTGCGGTGCTGCAACACTGTCTGCTGAACGCGTAAGAGGAGATAATAAGGAGTACGAGTATTCCGTAGAATGCCAAGAGGACAGTGCTTGTCCTGCGGCCGAGGAGTCTCTCCCAATAGAAGTGATGGTGGACGCGGTGCATAAACTGAAATATGAGAACTACACAAGCAGTTTTTTTATAAGAGATATCATCAAGCCCGATCCGCCGAAGAATTTGCAACTTAAACCGCTTAAAAACTCACGCCAGGTTGAAGTATCCTGGGAGTATCCGGATACATGGTCAACACCACACAGCTATTTTTCCCTTACCTTCTGTGTGCAGGTCCAAGGGAAGAGCAAAAGGGAGAAGAAGGACAGGGTATTCACTGATAAAACTTCCGCGACGGTCATCTGCCGAAAAAACGCTAGTATATCTGTACGGGCGCAGGATAGGTACTATAGTTCTTCTTGGTCTGAGTGGGCCTCAGTTCCGTGCTCTGGGGGAGGAAGTGGAGGAGGGTCCGGCGGTGGAAGCGGGGGAGGGAGTCGCAACTTGCCAGTGGCTACACCAGATCCAGGCATGTTTCCATGTCTGCATCATTCCCAGAATCTCCTGAGAGCGGTGTCAAATATGCTCCAAAAAGCGAGACAAACACTGGAATTTTACCCGTGTACCAGTGAGGAGATTGATCACGAGGACATAACCAAGGACAAGACCTCAACTGTAGAAGCGTGTTTGCCGCTGGAGTTGACTAAGAATGAGTCCTGCCTCAATTCCAGAGAAACTTCATTCATTACTAACGGCAGTTGTCTTGCATCCCGGAAAACGTCCTTTATGATGGCCCTTTGCCTTAGTTCAATTTACGAGGATCTTAAAATGTATCAAGTGGAGTTTAAAACCATGAATGCTAAACTTCTTATGGACCCCAAACGACAAATTTTTCTGGATCAGAATATGCTTGCCGTGATAGACGAACTCATGCAGGCGCTTAATTTTAACTCCGAAACAGTTCCACAAAAATCTAGCCTTGAAGAACCTGATTTTTATAAAACGAAGATTAAACTGTGTATCCTGCTGCATGCCTTTCGCATCCGAGCTGTCACAATCGATAGGGTTATGTCCTA CCTTAACGCGAGCtaGIL12p70 (Human; codon optimized; bold denotes signal sequence)(SEQ ID NO: 57) ATGTGCCATCAGCAACTCGTCATCTCCTGGTTCTCCCTTGTGTTCCTCGCTTCCCCTCTGGTCGCCATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAGCTGGATTGGTACCCGGACGCCCCTGGAGAAATGGTCGTGCTGACTTGCGATACGCCAGAAGAGGACGGCATAACCTGGACCCTGGATCAGAGCTCCGAGGTGCTCGGAAGCGGAAAGACCCTGACCATTCAAGTCAAGGAGTTCGGCGACGCGGGCCAGTACACTTGCCACAAGGGTGGCGAAGTGCTGTCCCACTCCCTGCTGCTGCTGCACAAGAAAGAGGATGGAATCTGGTCCACTGACATCCTCAAGGACCAAAAAGAACCGAAGAACAAGACCTTCCTCCGCTGCGAAGCCAAGAACTACAGCGGTCGGTTCACCTGTTGGTGGCTGACGACAATCTCCACCGACCTGACTTTCTCCGTGAAGTCGTCACGGGGATCAAGCGATCCTCAGGGCGTGACCTGTGGAGCCGCCACTCTGTCCGCCGAGAGAGTCAGGGGAGACAACAAGGAATATGAGTACTCCGTGGAATGCCAGGAGGACAGCGCCTGCCCTGCCGCGGAAGAGTCCCTGCCTATCGAGGTCATGGTCGATGCCGTGCATAAGCTGAAATACGAGAACTACACTTCCTCCTTCTTTATCCGCGACATCATCAAGCCTGACCCCCCCAAGAACTTGCAGCTGAAGCCACTCAAGAACTCCCGCCAAGTGGAAGTGTCTTGGGAATATCCAGACACTTGGAGCACCCCGCACTCATACTTCTCGCTCACTTTCTGTGTGCAAGTGCAGGGAAAGTCCAAACGGGAGAAGAAAGACCGGGTGTTCACCGACAAAACCTCCGCCACTGTGATTTGTCGGAAGAACGCGTCAATCAGCGTCCGGGCGCAGGATAGATACTACTCGTCCTCCTGGAGCGAATGGGCCAGCGTGCCTTGTTCCGGTGGCGGATCAGGCGGAGGTTCAGGAGGAGGCTCCGGAGGAGGTTCCCGGAACCTCCCTGTGGCAACCCCCGACCCTGGAATGTTCCCGTGCCTACACCACTCCCAAAACCTCCTGAGGGCTGTGTCGAACATGTTGCAGAAGGCCCGCCAGACCCTTGAGTTCTACCCCTGCACCTCGGAAGAAATTGATCACGAGGACATCACCAAGGACAAGACCTCGACCGTGGAAGCCTGCCTGCCGCTGGAACTGACCAAGAACGAATCGTGTCTGAACTCCCGCGAGACAAGCTTTATCACTAACGGCAGCTGCCTGGCGTCGAGAAAGACCTCATTCATGATGGCGCTCTGTCTTTCCTCGATCTACGAAGATCTGAAGATGTATCAGGTCGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCGAAGCGGCAGATCTTCCTGGACCAGAATATGCTCGCCGTGATTGATGAACTGATGCAGGCCCTGAATTTCAACTCCGAGACTGTGCCTCAAAAGTCCAGCCTGGAAGAACCGGACTTCTACAAGACCAAGATCAAGCTGTGCATCCTGTTGCACGCTTTCCGCATTCGAGCCGTGACCATTGACCGCGTGATGTCCTA CCTGAACGCCAGTIL12 (Mouse) (SEQ ID NO: 58)ATGTGTCCACAGAAGCTGACAATAAGTTGGTTTGCCATTGTCCTCCTGGTGAGCCCACTCATGGCAATGTGGGAACTCGAAAAGGATGTCTACGTGGTAGAAGTAGATTGGACTCCAGACGCGCCAGGGGAGACAGTGAATTTGACATGTGACACACCAGAAGAAGATGACATTACATGGACATCTGACCAACGCCATGGCGTAATAGGGAGTGGGAAAACACTCACGATCACAGTTAAAGAGTTCTTGGATGCTGGTCAATATACTTGCCATAAAGGCGGCGAGACACTCAGCCACTCACATTTGCTTTTGCATAAAAAAGAGAATGGCATTTGGAGCACTGAAATACTTAAGAACTTTAAGAACAAGACATTTCTCAAGTGTGAGGCCCCTAATTACAGCGGCAGGTTCACGTGCTCATGGCTGGTCCAGCGCAACATGGACCTCAAGTTTAACATAAAATCTTCTTCCTCTTCACCTGACTCCAGAGCTGTTACTTGCGGCATGGCTTCTCTGAGCGCAGAAAAAGTAACGTTGGATCAAAGAGACTACGAAAAGTACTCTGTTTCTTGTCAAGAGGATGTTACGTGCCCGACGGCCGAAGAAACGCTTCCAATTGAACTCGCGTTGGAAGCTCGCCAACAAAACAAGTATGAAAACTACAGTACAAGCTTCTTTATACGGGATATAATTAAACCCGATCCCCCCAAGAACTTGCAAATGAAACCACTTAAGAACAGCCAGGTGGAAGTTTCCTGGGAGTATCCAGACTCATGGAGTACTCCTCACAGCTATTTTTCTCTGAAATTCTTTGTAAGGATACAACGGAAGAAAGAGAAGATGAAAGAGACCGAGGAGGGTTGTAATCAGAAGGGAGCGTTTCTCGTGGAGAAAACGTCTACCGAAGTCCAATGTAAAGGTGGCAATGTGTGCGTCCAAGCTCAGGATAGATACTATAATTCAAGTTGCTCCAAGTGGGCCTGTGTTCCATGCCGCGTTCGGAGCGGGGGAGGTAGCGGAGGAGGTAGTGGGGGTGGGTCAGGAGGAGGGAGTCGAGTTATCCCGGTGTCAGGCCCCGCACGCTGCTTGAGCCAGAGTCGCAACCTCCTTAAGACAACAGATGACATGGTGAAAACAGCACGCGAAAAGCTTAAACACTACTCTTGTACGGCGGAGGATATTGATCACGAGGATATTACCCGAGACCAAACTAGCACTTTGAAAACCTGTCTGCCCCTTGAACTTCATAAAAATGAGAGCTGTCTGGCTACACGAGAGACGTCAAGTACGACTAGGGGCAGCTGTCTCCCGCCGCAAAAGACAAGCCTCATGATGACGCTCTGTTTGGGTTCCATTTACGAGGACTTGAAAATGTATCAAACGGAGTTCCAGGCTATAAATGCGGCGTTGCAGAACCATAACCATCAACAAATTATACTTGATAAAGGCATGTTGGTGGCGATTGATGAACTCATGCAGAGTCTCAATCACAACGGGGAAACGTTGAGACAGAAACCCCCAGTCGGTGAAGCGGACCCATATCGAGTAAAAATGAAGCTCTGCATTCTGCTTCACGCATTCAGCACTAGAGTTGTTACCATCAACCGGGT AATGGGATATCTCTCCAGTGCGtaGIL21 (Human; codon optimized; bold denotes signal sequence)(SEQ ID NO: 59) ATGGAACGCATTGTGATCTGCCTGATGGTCATCTTCCTGGGCACCTTAGTGCACAAGTCGAGCAGCCAGGGACAGGACAGGCACATGATTAGAATGCGCCAGCTCATCGATATCGTGGACCAGTTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCGGCCCCCGAAGATGTGGAAACCAATTGCGAATGGTCGGCATTTTCCTGCTTTCAAAAGGCACAGCTCAAGTCCGCTAACACCGGGAACAACGAACGGATCATCAACGTGTCCATCAAAAAGCTGAAGCGGAAGCCTCCCTCCACCAACGCCGGACGGAGGCAGAAGCATAGGCTGACTTGCCCGTCATGCGACTCCTACGAGAAGAAGCCGCCGAAGGAGTTCCTGGAGCGGTTCAAGTCGCTCCTGCAAAAGATGATTCATCAGCACCTGTCCTCCCGGACTCATGGGTCTGAGG ATTCA IL12p70_T2A_IL21(Human; codon optimized; bold denotes signal sequences) (SEQ ID NO: 60)ATGTGCCATCAGCAACTCGTCATCTCCTGGTTCTCCCTTGTGTTCCTCGCTTCCCCTCTGGTCGCCATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAGCTGGATTGGTACCCGGACGCCCCTGGAGAAATGGTCGTGCTGACTTGCGATACGCCAGAAGAGGACGGCATAACCTGGACCCTGGATCAGAGCTCCGAGGTGCTCGGAAGCGGAAAGACCCTGACCATTCAAGTCAAGGAGTTCGGCGACGCGGGCCAGTACACTTGCCACAAGGGTGGCGAAGTGCTGTCCCACTCCCTGCTGCTGCTGCACAAGAAAGAGGATGGAATCTGGTCCACTGACATCCTCAAGGACCAAAAAGAACCGAAGAACAAGACCTTCCTCCGCTGCGAAGCCAAGAACTACAGCGGTCGGTTCACCTGTTGGTGGCTGACGACAATCTCCACCGACCTGACTTTCTCCGTGAAGTCGTCACGGGGATCAAGCGATCCTCAGGGCGTGACCTGTGGAGCCGCCACTCTGTCCGCCGAGAGAGTCAGGGGAGACAACAAGGAATATGAGTACTCCGTGGAATGCCAGGAGGACAGCGCCTGCCCTGCCGCGGAAGAGTCCCTGCCTATCGAGGTCATGGTCGATGCCGTGCATAAGCTGAAATACGAGAACTACACTTCCTCCTTCTTTATCCGCGACATCATCAAGCCTGACCCCCCCAAGAACTTGCAGCTGAAGCCACTCAAGAACTCCCGCCAAGTGGAAGTGTCTTGGGAATATCCAGACACTTGGAGCACCCCGCACTCATACTTCTCGCTCACTTTCTGTGTGCAAGTGCAGGGAAAGTCCAAACGGGAGAAGAAAGACCGGGTGTTCACCGACAAAACCTCCGCCACTGTGATTTGTCGGAAGAACGCGTCAATCAGCGTCCGGGCGCAGGATAGATACTACTCGTCCTCCTGGAGCGAATGGGCCAGCGTGCCTTGTTCCGGTGGCGGATCAGGCGGAGGTTCAGGAGGAGGCTCCGGAGGAGGTTCCCGGAACCTCCCTGTGGCAACCCCCGACCCTGGAATGTTCCCGTGCCTACACCACTCCCAAAACCTCCTGAGGGCTGTGTCGAACATGTTGCAGAAGGCCCGCCAGACCCTTGAGTTCTACCCCTGCACCTCGGAAGAAATTGATCACGAGGACATCACCAAGGACAAGACCTCGACCGTGGAAGCCTGCCTGCCGCTGGAACTGACCAAGAACGAATCGTGTCTGAACTCCCGCGAGACAAGCTTTATCACTAACGGCAGCTGCCTGGCGTCGAGAAAGACCTCATTCATGATGGCGCTCTGTCTTTCCTCGATCTACGAAGATCTGAAGATGTATCAGGTCGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCGAAGCGGCAGATCTTCCTGGACCAGAATATGCTCGCCGTGATTGATGAACTGATGCAGGCCCTGAATTTCAACTCCGAGACTGTGCCTCAAAAGTCCAGCCTGGAAGAACCGGACTTCTACAAGACCAAGATCAAGCTGTGCATCCTGTTGCACGCTTTCCGCATTCGAGCCGTGACCATTGACCGCGTGATGTCCTACCTGAACGCCAGTAGACGGAAACGCGGAAGCGGAGAGGGCAGAGGCTCGCTGCTTACATGCGGGGACGTGGAAGAGAACCCCGGTCCGATGGAACGCATTGTGATCTGCCTGATGGTCATCTTCCTGGGCACCTTAGTGCACAAGTCGAGCAGCCAGGGACAGGACAGGCACATGATTAGAATGCGCCAGCTCATCGATATCGTGGACCAGTTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCGGCCCCCGAAGATGTGGAAACCAATTGCGAATGGTCGGCATTTTCCTGCTTTCAAAAGGCACAGCTCAAGTCCGCTAACACCGGGAACAACGAACGGATCATCAACGTGTCCATCAAAAAGCTGAAGCGGAAGCCTCCCTCCACCAACGCCGGACGGAGGCAGAAGCATAGGCTGACTTGCCCGTCATGCGACTCCTACGAGAAGAAGCCGCCGAAGGAGTTCCTGGAGCGGTTCAAGTCGCTCCTGCAAAAGATGATTCATCAGCACCTGTCCTCCCGGACTCATGGGTCTGAGGATTC AIL-12 2A CCL21a (Human) (SEQ ID NO: 61)ATGTGCCATCAGCAGCTTGTCATATCTTGGTTTTCACTTGTATTCCTGGCCAGCCCTTTGGTTGCGATCTGGGAGCTCAAGAAGGATGTGTACGTTGTAGAGCTGGACTGGTACCCCGATGCTCCCGGTGAGATGGTCGTTTTGACATGTGACACTCCAGAAGAGGACGGTATTACGTGGACTCTGGACCAGTCCTCCGAAGTTCTTGGTTCTGGTAAGACTCTGACTATCCAGGTGAAAGAATTTGGGGATGCGGGACAATACACATGCCACAAGGGAGGCGAGGTGTTGTCTCATAGTTTGCTGCTTCTCCACAAGAAAGAGGATGGAATCTGGAGCACCGACATACTCAAGGATCAAAAGGAACCCAAAAATAAGACATTTCTGCGATGTGAGGCTAAGAACTATAGTGGCCGCTTCACTTGTTGGTGGCTGACTACCATCAGCACAGATCTCACGTTTTCAGTAAAAAGTAGTAGAGGTTCAAGTGATCCTCAAGGGGTAACGTGCGGTGCTGCAACACTGTCTGCTGAACGCGTAAGAGGAGATAATAAGGAGTACGAGTATTCCGTAGAATGCCAAGAGGACAGTGCTTGTCCTGCGGCCGAGGAGTCTCTCCCAATAGAAGTGATGGTGGACGCGGTGCATAAACTGAAATATGAGAACTACACAAGCAGTTTTTTTATAAGAGATATCATCAAGCCCGATCCGCCGAAGAATTTGCAACTTAAACCGCTTAAAAACTCACGCCAGGTTGAAGTATCCTGGGAGTATCCGGATACATGGTCAACACCACACAGCTATTTTTCCCTTACCTTCTGTGTGCAGGTCCAAGGGAAGAGCAAAAGGGAGAAGAAGGACAGGGTATTCACTGATAAAACTTCCGCGACGGTCATCTGCCGAAAAAACGCTAGTATATCTGTACGGGCGCAGGATAGGTACTATAGTTCTTCTTGGTCTGAGTGGGCCTCAGTTCCGTGCTCTGGGGGAGGAAGTGGAGGAGGGTCCGGCGGTGGAAGCGGGGGAGGGAGTCGCAACTTGCCAGTGGCTACACCAGATCCAGGCATGTTTCCATGTCTGCATCATTCCCAGAATCTCCTGAGAGCGGTGTCAAATATGCTCCAAAAAGCGAGACAAACACTGGAATTTTACCCGTGTACCAGTGAGGAGATTGATCACGAGGACATAACCAAGGACAAGACCTCAACTGTAGAAGCGTGTTTGCCGCTGGAGTTGACTAAGAATGAGTCCTGCCTCAATTCCAGAGAAACTTCATTCATTACTAACGGCAGTTGTCTTGCATCCCGGAAAACGTCCTTTATGATGGCCCTTTGCCTTAGTTCAATTTACGAGGATCTTAAAATGTATCAAGTGGAGTTTAAAACCATGAATGCTAAACTTCTTATGGACCCCAAACGACAAATTTTTCTGGATCAGAATATGCTTGCCGTGATAGACGAACTCATGCAGGCGCTTAATTTTAACTCCGAAACAGTTCCACAAAAATCTAGCCTTGAAGAACCTGATTTTTATAAAACGAAGATTAAACTGTGTATCCTGCTGCATGCCTTTCGCATCCGAGCTGTCACAATCGATAGGGTTATGTCCTACCTTAACGCGAGCCGGCGCAAGAGGGGTTCCGGAGAGGGAAGGGGTAGTCTGCTCACCTGCGGCGATGTTGAAGAAAATCCTGGTCCCATGGCGCAAAGTCTGGCTCTTTCACTCCTGATCCTGGTCTTGGCCTTCGGGATTCCGAGGACCCAAGGAAGTGATGGTGGCGCCCAAGATTGTTGCCTTAAATACAGCCAGCGGAAAATACCCGCGAAAGTGGTCAGGAGTTATAGAAAACAGGAGCCTTCCCTGGGTTGTAGTATCCCCGCCATACTTTTCCTCCCGAGAAAACGGAGCCAGGCCGAACTGTGCGCTGACCCTAAGGAACTTTGGGTGCAACAACTTATGCAACACCTGGATAAGACACCTTCTCCTCAAAAGCCAGCTCAGGGCTGCCGAAAAGATAGAGGCGCCTCAAAAACCGGAAAAAAGGGCAAAGGTTCTAAAGGATGTAAGCGGACTGAACGCTCTCAAACGCCTAAAGGGCCGtaGIL-12 2A CCL21a (Mouse) (SEQ ID NO: 62)ATGTGTCCACAGAAGCTGACAATAAGTTGGTTTGCCATTGTCCTCCTGGTGAGCCCACTCATGGCAATGTGGGAACTCGAAAAGGATGTCTACGTGGTAGAAGTAGATTGGACTCCAGACGCGCCAGGGGAGACAGTGAATTTGACATGTGACACACCAGAAGAAGATGACATTACATGGACATCTGACCAACGCCATGGCGTAATAGGGAGTGGGAAAACACTCACGATCACAGTTAAAGAGTTCTTGGATGCTGGTCAATATACTTGCCATAAAGGCGGCGAGACACTCAGCCACTCACATTTGCTTTTGCATAAAAAAGAGAATGGCATTTGGAGCACTGAAATACTTAAGAACTTTAAGAACAAGACATTTCTCAAGTGTGAGGCCCCTAATTACAGCGGCAGGTTCACGTGCTCATGGCTGGTCCAGCGCAACATGGACCTCAAGTTTAACATAAAATCTTCTTCCTCTTCACCTGACTCCAGAGCTGTTACTTGCGGCATGGCTTCTCTGAGCGCAGAAAAAGTAACGTTGGATCAAAGAGACTACGAAAAGTACTCTGTTTCTTGTCAAGAGGATGTTACGTGCCCGACGGCCGAAGAAACGCTTCCAATTGAACTCGCGTTGGAAGCTCGCCAACAAAACAAGTATGAAAACTACAGTACAAGCTTCTTTATACGGGATATAATTAAACCCGATCCCCCCAAGAACTTGCAAATGAAACCACTTAAGAACAGCCAGGTGGAAGTTTCCTGGGAGTATCCAGACTCATGGAGTACTCCTCACAGCTATTTTTCTCTGAAATTCTTTGTAAGGATACAACGGAAGAAAGAGAAGATGAAAGAGACCGAGGAGGGTTGTAATCAGAAGGGAGCGTTTCTCGTGGAGAAAACGTCTACCGAAGTCCAATGTAAAGGTGGCAATGTGTGCGTCCAAGCTCAGGATAGATACTATAATTCAAGTTGCTCCAAGTGGGCCTGTGTTCCATGCCGCGTTCGGAGCGGGGGAGGTAGCGGAGGAGGTAGTGGGGGTGGGTCAGGAGGAGGGAGTCGAGTTATCCCGGTGTCAGGCCCCGCACGCTGCTTGAGCCAGAGTCGCAACCTCCTTAAGACAACAGATGACATGGTGAAAACAGCACGCGAAAAGCTTAAACACTACTCTTGTACGGCGGAGGATATTGATCACGAGGATATTACCCGAGACCAAACTAGCACTTTGAAAACCTGTCTGCCCCTTGAACTTCATAAAAATGAGAGCTGTCTGGCTACACGAGAGACGTCAAGTACGACTAGGGGCAGCTGTCTCCCGCCGCAAAAGACAAGCCTCATGATGACGCTCTGTTTGGGTTCCATTTACGAGGACTTGAAAATGTATCAAACGGAGTTCCAGGCTATAAATGCGGCGTTGCAGAACCATAACCATCAACAAATTATACTTGATAAAGGCATGTTGGTGGCGATTGATGAACTCATGCAGAGTCTCAATCACAACGGGGAAACGTTGAGACAGAAACCCCCAGTCGGTGAAGCGGACCCATATCGAGTAAAAATGAAGCTCTGCATTCTGCTTCACGCATTCAGCACTAGAGTTGTTACCATCAACCGGGTAATGGGATATCTCTCCAGTGCGCGGCGCAAGAGGGGTTCCGGAGAGGGAAGGGGTAGTCTGCTCACCTGCGGCGATGTTGAAGAAAATCCTGGTCCCATGGCGCAAATGATGACCCTTTCCCTGCTGAGTCTTGTCCTCGCGCTCTGCATCCCGTGGACGCAGGGGTCTGATGGGGGGGGCCAAGACTGTTGCCTGAAGTATTCACAAAAAAAGATACCGTACTCTATTGTCAGAGGGTACAGGAAGCAAGAACCCTCCTTGGGTTGCCCTATACCAGCAATTCTTTTCTCCCCACGCAAGCATTCCAAACCAGAACTGTGTGCGAACCCCGAGGAGGGTTGGGTACAGAACTTGATGCGAAGGCTTGACCAGCCCCCAGCCCCTGGCAAGCAGTCACCTGGGTGCAGAAAAAACAGAGGTACTTCAAAGAGCGGCAAGAAAGGCAAAGGGAGTAAAGGATGTAAAAGAACGGAGCAGACCCAGCCTTCACGAGGCtaGCCL21a 2A IL-12 (Mouse) (SEQ ID NO: 63)ATGGCGCAAATGATGACCCTTTCCCTGCTGAGTCTTGTCCTCGCGCTCTGCATCCCGTGGACGCAGGGGTCTGATGGGGGGGGCCAAGACTGTTGCCTGAAGTATTCACAAAAAAAGATACCGTACTCTATTGTCAGAGGGTACAGGAAGCAAGAACCCTCCTTGGGTTGCCCTATACCAGCAATTCTTTTCTCCCCACGCAAGCATTCCAAACCAGAACTGTGTGCGAACCCCGAGGAGGGTTGGGTACAGAACTTGATGCGAAGGCTTGACCAGCCCCCAGCCCCTGGCAAGCAGTCACCTGGGTGCAGAAAAAACAGAGGTACTTCAAAGAGCGGCAAGAAAGGCAAAGGGAGTAAAGGATGTAAAAGAACGGAGCAGACCCAGCCTTCACGAGGCCGGCGCAAGAGGGGTTCCGGAGAGGGAAGGGGTAGTCTGCTCACCTGCGGCGATGTTGAAGAAAATCCTGGTCCCATGTGTCCACAGAAGCTGACAATAAGTTGGTTTGCCATTGTCCTCCTGGTGAGCCCACTCATGGCAATGTGGGAACTCGAAAAGGATGTCTACGTGGTAGAAGTAGATTGGACTCCAGACGCGCCAGGGGAGACAGTGAATTTGACATGTGACACACCAGAAGAAGATGACATTACATGGACATCTGACCAACGCCATGGCGTAATAGGGAGTGGGAAAACACTCACGATCACAGTTAAAGAGTTCTTGGATGCTGGTCAATATACTTGCCATAAAGGCGGCGAGACACTCAGCCACTCACATTTGCTTTTGCATAAAAAAGAGAATGGCATTTGGAGCACTGAAATACTTAAGAACTTTAAGAACAAGACATTTCTCAAGTGTGAGGCCCCTAATTACAGCGGCAGGTTCACGTGCTCATGGCTGGTCCAGCGCAACATGGACCTCAAGTTTAACATAAAATCTTCTTCCTCTTCACCTGACTCCAGAGCTGTTACTTGCGGCATGGCTTCTCTGAGCGCAGAAAAAGTAACGTTGGATCAAAGAGACTACGAAAAGTACTCTGTTTCTTGTCAAGAGGATGTTACGTGCCCGACGGCCGAAGAAACGCTTCCAATTGAACTCGCGTTGGAAGCTCGCCAACAAAACAAGTATGAAAACTACAGTACAAGCTTCTTTATACGGGATATAATTAAACCCGATCCCCCCAAGAACTTGCAAATGAAACCACTTAAGAACAGCCAGGTGGAAGTTTCCTGGGAGTATCCAGACTCATGGAGTACTCCTCACAGCTATTTTTCTCTGAAATTCTTTGTAAGGATACAACGGAAGAAAGAGAAGATGAAAGAGACCGAGGAGGGTTGTAATCAGAAGGGAGCGTTTCTCGTGGAGAAAACGTCTACCGAAGTCCAATGTAAAGGTGGCAATGTGTGCGTCCAAGCTCAGGATAGATACTATAATTCAAGTTGCTCCAAGTGGGCCTGTGTTCCATGCCGCGTTCGGAGCGGGGGAGGTAGCGGAGGAGGTAGTGGGGGTGGGTCAGGAGGAGGGAGTCGAGTTATCCCGGTGTCAGGCCCCGCACGCTGCTTGAGCCAGAGTCGCAACCTCCTTAAGACAACAGATGACATGGTGAAAACAGCACGCGAAAAGCTTAAACACTACTCTTGTACGGCGGAGGATATTGATCACGAGGATATTACCCGAGACCAAACTAGCACTTTGAAAACCTGTCTGCCCCTTGAACTTCATAAAAATGAGAGCTGTCTGGCTACACGAGAGACGTCAAGTACGACTAGGGGCAGCTGTCTCCCGCCGCAAAAGACAAGCCTCATGATGACGCTCTGTTTGGGTTCCATTTACGAGGACTTGAAAATGTATCAAACGGAGTTCCAGGCTATAAATGCGGCGTTGCAGAACCATAACCATCAACAAATTATACTTGATAAAGGCATGTTGGTGGCGATTGATGAACTCATGCAGAGTCTCAATCACAACGGGGAAACGTTGAGACAGAAACCCCCAGTCGGTGAAGCGGACCCATATCGAGTAAAAATGAAGCTCTGCATTCTGCTTCACGCATTCAGCACTAGAGTTGTTACCATCAACCGGGTAATGGGATATCTCTCCAGTGCGtaG IL7 (Mouse) (SEQ ID NO: 64)ATGTTTCATGTGTCCTTCAGGTACATATTTGGTATCCCACCACTTATATTGGTGCTCTTGCCTGTAACCAGCTCTGAATGTCATATAAAAGACAAGGAGGGCAAAGCATACGAGTCCGTATTGATGATCTCAATCGATGAACTTGACAAGATGACAGGGACCGATTCTAATTGTCCAAATAACGAGCCAAACTTCTTTCGGAAACACGTGTGTGATGATACAAAAGAAGCTGCTTTTCTTAACAGAGCTGCCAGAAAACTCAAGCAGTTCCTCAAGATGAATATATCCGAGGAATTTAACGTGCATCTCCTCACAGTATCTCAGGGAACTCAAACCCTTGTAAACTGCACTTCTAAGGAGGAGAAGAATGTCAAAGAGCAGAAGAAAAATGATGCATGTTTTTTGAAACGGCTGTTGAGGGAGATCAAAACATGCTGGAATAAAATCCTCAAGGGCTCAA TTtaGIL15 (Human) (SEQ ID NO: 65)ATGGAAACAGACACATTGCTGCTTTGGGTATTGTTGCTCTGGGTGCCTGGATCAACAGGAAACTGGGTAAACGTAATTTCAGATCTGAAGAAGATCGAGGACCTTATTCAATCCATGCACATCGATGCCACTCTCTACACCGAAAGCGACGTTCACCCATCTTGCAAGGTGACCGCTATGAAATGTGAATTGTTGGAACTTCAGGTAATTTCTCTGGAGAGCGGCGATGCCTCAATACATGACACCGTTGAAAATCTTATCATCCTTGCTAATGATTCACTCTCTAGTAATGGGAACGTAACAGAGAGCGGGTGTAAGGAGTGTGAAGAACTGGAGGAGAAAAACATTAAGGAATTTTTGCAGTCATTCGTCCATATAGTGCAAATGTTCATAAACACTTCCAGAAGAAAGCGAGGCTCTGGGGAGGGGCGAGGCTCTCTGCTGACCTGTGGGGATGTAGAAGAGAATCCAGGTCCCATGGACCGGCTGACCAGCTCATTCCTGCTTCTGATTGTGCCAGCCTACGTGCTCTCCATCACATGTCCTCCCCCAATGAGCGTCGAGCATGCTGACATCTGGGTGAAGTCATACTCCTTGTACAGCAGAGAGAGATACATTTGTAATTCCGGATTCAAGCGCAAGGCCGGCACCTCCTCTCTGACAGAGTGCGTCCTTAACAAAGCAACCAACGTAGCACATTGGACCACACCATCCTTGAAGTGCATACGAGAACCTAAATCTTGCGATAAGACTCATACTTGTCCACCTTGTCCAGCCCCAGAACTGCTTGGCGGACCCTCAGTATTTTTGTTCCCACCAAAGCCAAAAGACACACTCATGATATCCAGAACTCCTGAGGTGACCTGTGTCGTTGTAGACGTTTCCCACGAAGATCCTGAAGTAAAATTCAACTGGTACGTGGATGGGGTCGAAGTCCATAACGCCAAGACTAAACCAAGGGAGGAACAGTATAACTCTACTTACCGAGTAGTTTCTGTGTTGACCGTGCTGCACCAGGACTGGTTGAACGGGAAGGAGTACAAATGCAAGGTGAGCAATAAAGCTCTGCCCGCACCAATCGAAAAGACAATATCTAAGGCCAAGGGGCAGCCACGAGAGCCCCAGGTATACACACTGCCACCCTCACGCGATGAATTGACTAAGAACCAGGTTTCCCTGACCTGTCTTGTAAAAGGTTTCTACCCTTCCGACATAGCTGTTGAGTGGGAAAGTAACGGGCAGCCAGAGAACAATTACAAGACAACTCCACCCGTTCTTGATAGCGATGGATCATTTTTTCTGTATTCCAAACTCACTGTCGATAAAAGTCGCTGGCAGCAAGGCAATGTTTTTAGCTGCTCAGTCATGCACGAAGCACTGCATAATCACTACACACAAAAAAGTTTGTCCCTTAGCCCTGGTAAGt aGIL15 (Human) (SEQ ID NO: 66)ATGTACTCAATGCAGTTGGCCTCCTGTGTAACATTGACCTTGGTCCTCTTGGTCAACAGCAATTGGATCGATGTACGCTACGACTTGGAGAAGATTGAGTCCCTTATACAGAGTATACACATAGATACAACCTTGTATACTGACAGTGACTTCCATCCCAGCTGTAAAGTGACTGCAATGAACTGTTTTTTGTTGGAGTTGCAAGTAATTCTGCATGAATACAGCAACATGACCCTCAATGAAACCGTTAGGAATGTCCTTTATCTCGCAAATTCTACTCTGAGTAGCAATAAGAATGTTGCCGAAAGCGGCTGCAAGGAGTGCGAAGAACTGGAGGAAAAAACTTTCACCGAGTTTCTCCAGAGTTTCATCAGAATTGTCCAAATGTTCATTAATACAAGTAGTGGTGGTGGGAGCGGGGGTGGAGGCAGTGGGGGAGGTGGGAGCGGAGGTGGAGGGTCCGGAGGGGGGAGCCTTCAAGGCACTACTTGTCCTCCACCCGTATCCATCGAGCACGCCGATATTCGAGTTAAAAATTATAGTGTTAATAGCAGAGAACGATACGTCTGCAACTCAGGGTTTAAGAGAAAGGCCGGAACTTCAACTCTCATAGAATGCGTGATTAATAAGAATACTAACGTCGCACATTGGACTACTCCCAGTCTCAAGTGCATACGCGATCCATCTCTCGCTCATTACTCACCAGTACCTACAGTGGTTACTCCTAAGGTGACCTCTCAGCCCGAATCACCATCTCCCAGCGCAAAAGAGCCTGAGGCCTTTTCTCCTAAATCAGACACTGCTATGACTACAGAAACAGCCATAATGCCAGGAAGCCGGCTGACACCATCTCAAACTACCAGCGCAGGCACAACTGGGACTGGCTCCCACAAAAGCTCACGCGCACCAAGTCTCGCCGCAACAATGACATTGGAGCCTACAGCCAGCACATCTCTTAGAATCACAGAAATTTCTCCCCACAGTAGCAAGATGACCAAGGTGGCAATTAGTACCAGCGTCCTTCTTGTAGGAGCTGGAGTTGTGATGGCATTTTTGGC ATGGTATATCAAAAGCAGGtaGIL15 (Mouse) (SEQ ID NO: 67)ATGAAGATCCTCAAGCCATACATGCGAAACACTAGTATTAGCTGTTACTTGTGTTTTCTGCTGAATAGTCATTTTTTGACTGAAGCAGGAATCCATGTATTTATACTCGGTTGTGTGTCTGTAGGTCTGCCAAAGACTGAGGCTAATTGGATTGACGTGCGCTATGATCTTGAAAAAATAGAGTCCTTGATTCAATCAATACACATCGATACCACTCTCTACACCGACAGTGATTTCCATCCTTCCTGCAAGGTAACAGCTATGAATTGCTTCCTCCTGGAGCTCCAAGTCATTCTCCATGAGTACTCCAACATGACTTTGAACGAAACTGTAAGAAACGTATTGTATCTGGCTAATAGCACCTTGTCTAGTAACAAAAATGTGGCAGAGAGCGGCTGCAAAGAATGTGAAGAATTGGAAGAGAAAACATTTACAGAGTTCCTGCAATCCTTTATTCGCATCGTCCAAATGTTTATCAATACCTCTtaG IL15 (Mouse) (SEQ ID NO: 68)ATGTATTCCATGCAACTTGCCAGTTGTGTAACCCTTACTCTCGTCCTGCTCGTTAATTCCGCTGGTGCTAACTGGATAGATGTTCGATACGATCTGGAAAAGATTGAGTCCCTTATCCAATCCATTCATATAGATACCACCCTTTATACTGACAGCGACTTCCATCCTTCTTGCAAGGTGACCGCTATGAATTGTTTCCTGCTGGAACTCCAAGTTATTCTGCATGAATACTCTAATATGACACTTAACGAGACCGTAAGAAATGTTCTCTATCTCGCTAATAGTACTTTGAGCTCAAATAAGAACGTGGCCGAGTCTGGGTGTAAGGAATGCGAAGAGCTGGAAGAAAAGACATTCACCGAGTTTCTCCAGTCTTTCATACGGATTGTGCAGATGTTTATCAACACATCAGATTACAAAGACGACGATGATAAGtaG IL18 (Mouse) (SEQ ID NO: 69)ATGGCAGCCATGTCTGAGGACTCTTGTGTGAACTTTAAAGAAATGATGTTCATAGACAATACACTCTACTTTATACCTGAGGAGAATGGAGATTTGGAATCTGACAACTTTGGCAGGCTGCATTGCACTACCGCAGTTATCCGAAACATCAACGATCAGGTACTGTTTGTTGATAAAAGACAACCTGTATTCGAGGACATGACCGACATAGATCAGTCTGCCTCAGAGCCCCAGACTAGGCTTATCATCTATATGTACAAGGACAGCGAAGTACGAGGCCTGGCTGTTACACTCTCAGTCAAAGACTCTAAGATGAGCACCCTGTCATGCAAGAACAAAATTATCAGTTTTGAGGAGATGGACCCACCTGAAAACATAGATGACATTCAGTCAGACCTCATTTTTTTTCAAAAGCGGGTACCAGGACACAACAAAATGGAATTTGAATCATCACTCTACGAAGGACATTTCCTTGCATGCCAGAAAGAGGATGACGCATTCAAATTGATCCTGAAAAAAAAGGACGAAAATGGTGATAAATCAGTCATGTTTACATTGACCAATCTTCACCAAAGTtaG IL18 (Mouse) (SEQ ID NO: 70)ATGGCTGCAATGTCTGAAGATAGCTGTGTCAACTTTAAGGAGATGATGTTCATTGATAATACTTTGTACTTTATACCTGAAGAAAATGGAGACCTTGAGTCAGACAACTTCGGGAGACTGCACTGCACAACTGCCGTTATCCGAAACATAAATGATCAAGTATTGTTCGTGGACAAAAGACAACCAGTCTTTGAGGATATGACAGACATCGACCAATCCGCATCTGAACCTCAGACTAGGCTGATCATCTATATGTACGCCGACTCCGAAGTAAGAGGCCTTGCTGTGACACTTAGTGTTAAGGATAGTAAGATGAGCACACTGTCCTGTAAGAATAAGATTATATCTTTTGAAGAGATGGACCCTCCCGAGAACATAGATGACATCCAGAGCGACTTGATCTTCTTTCAGAAGCGAGTGCCAGGCCATAACAAGATGGAATTTGAATCATCTCTTTATGAAGGCCATTTCCTCGCATGTCAAAAGGAGGACGATGCCTTCAAGCTCATTCTGAAAAAAAAAGACGAGAACGGTGATAAGAGCGTGATGTTCACTCTGACAAATCTGCACCAGTCAtaG IL18 (Human) (SEQ ID NO: 71)ATGTATCGCATGCAACTCCTGTCCTGCATTGCTCTGAGCTTGGCTTTGGTAACCAACTCATACTTCGGGAAACTGGAGAGTAAACTCTCCGTAATCAGGAATCTTAATGACCAAGTATTGTTTATTGACCAGGGCAACCGCCCGTTGTTCGAGGATATGACTGATTCTGACTGTCGGGATAACGCTCCGAGAACTATCTTTATCATTTCAATGTACAAGGACAGCCAACCGCGGGGTATGGCTGTGACAATCAGTGTCAAATGTGAGAAGATTTCCACGCTGTCCTGCGAAAACAAGATAATTTCTTTCAAAGAAATGAACCCCCCTGACAATATAAAGGATACAAAGAGTGATATCATCTTCTTTCAGAGGTCCGTGCCCGGCCACGATAATAAGATGCAATTTGAAAGTTCATCTTATGAGGGGTACTTTTTGGCATGCGAGAAAGAAAGGGATCTCTTCAAGTTGATCCTGAAGAAGGAGGACGAATTGGGCGACCGCTCCATCATGTTCACAGTCCAGAACGAGGACtaG IL18 (Human) (SEQ ID NO: 72)ATGTACCGCATGCAGCTCCTGAGTTGTATTGCCCTTTCCCTCGCTCTCGTTACCAATTCTTACTTCGGTAAGCTTGCCTCTAAACTCTCTGTTATTAGGAACTTGAACGACCAAGTCCTTTTCATAGACCAAGGGAACAGACCACTGTTTGAAGATATGACGGATAGCGATTGCCGAGATAATGCCCCTAGGACGATTTTTATCATTAGTATGTATGCGGACTCTCAACCGAGGGGGATGGCCGTTACTATAAGTGTGAAATGCGAGAAAATATCAACGCTCAGTTGTGAGAACAAAATCATAAGTTTCAAGGAGATGAATCCACCTGATAACATCAAAGACACTAAGTCTGATATTATATTTTTCCAACGAAGTGTTCCGGGACACGATAACAAAATGCAATTTGAGAGCTCCTCATACGAGGGCTACTTCCTCGCGTGTGAGAAAGAAAGGGATTTGTTTAAGCTTATCCTCAAGAAAGAGGACGAGTTGGGGGATCGGAGCATAATGTTTACCGTACAGAATGAGGACtaG IL21 (Mouse) (SEQ ID NO: 73)ATGGAGCGGACACTCGTGTGTCTTGTCGTAATTTTTCTCGGGACAGTCGCACACAAGTCCTCACCCCAGGGTCCTGATCGCCTTCTCATACGCCTCCGACATTTGATCGACATTGTAGAGCAGCTCAAAATTTACGAGAATGACCTCGATCCCGAGCTTTTGAGTGCTCCCCAAGACGTTAAGGGTCATTGCGAGCACGCAGCTTTTGCTTGCTTCCAGAAGGCCAAGTTGAAACCAAGCAACCCTGGTAATAATAAGACTTTCATCATCGACTTGGTCGCCCAACTCCGAAGGAGGCTGCCTGCCCGGCGCGGAGGAAAAAAACAAAAGCATATTGCAAAGTGTCCTTCATGTGATTCATACGAAAAGCGGACTCCCAAAGAGTTCTTGGAAAGGTTGAAATGGCTTCTTCAGAAGATGATTCATCAACATTTGTCAtaG IFN-beta (Human) (SEQ ID NO: 74)ATGACCAACAAATGCCTTTTGCAAATTGCCCTGCTTTTGTGTTTTAGCACTACCGCATTGAGCATGTCATATAACCTCCTCGGCTTCCTTCAGAGATCATCAAACTTTCAGTGTCAGAAACTGCTTTGGCAACTTAATGGCAGGCTCGAATATTGTCTGAAAGATCGGATGAATTTCGACATTCCAGAAGAAATAAAACAGCTTCAACAATTCCAGAAAGAGGACGCCGCCCTGACTATTTACGAGATGCTCCAGAATATCTTCGCCATTTTCCGGCAGGACAGCTCATCCACGGGGTGGAATGAGACTATTGTAGAAAATCTTCTGGCTAATGTGTACCATCAAATTAATCACCTCAAAACGGTGCTTGAGGAAAAACTTGAAAAGGAAGATTTCACACGGGGCAAGTTGATGTCCTCCCTGCACCTTAAACGATACTACGGCAGGATTCTTCATTACTTGAAGGCTAAGGAGTATAGCCATTGCGCGTGGACAATTGTACGGGTAGAAATACTGCGAAACTTTTATTTCATCAACCGG CTCACTGGATACCTTAGAAATtaGIFN-beta (Mouse) (SEQ ID NO: 75)ATGAACAATCGGTGGATACTCCACGCCGCATTTCTCCTCTGCTTTAGCACGACGGCCCTGTCCATCAACTACAAACAGCTTCAGTTGCAGGAGCGGACTAACATAAGGAAGTGCCAGGAACTGCTGGAACAGCTTAATGGTAAAATTAATCTTACATACCGAGCTGACTTCAAAATTCCTATGGAAATGACCGAGAAGATGCAGAAATCCTACACGGCATTCGCCATCCAGGAAATGCTCCAGAACGTATTTCTCGTGTTCCGCAATAATTTCTCTTCTACGGGTTGGAACGAAACCATTGTTGTTAGACTGCTTGACGAACTGCATCAGCAAACCGTGTTCCTTAAAACCGTGCTTGAGGAGAAGCAGGAGGAGCGCCTGACTTGGGAGATGTCTAGTACCGCACTTCACTTGAAATCCTACTACTGGCGCGTTCAGCGGTATCTGAAGCTGATGAAGTATAACTCATACGCCTGGATGGTAGTGCGCGCAGAGATCTTCAGAAACTTTCTTATCATCCGGCGACTGACCCGAAACTTT CAGAATtaGIFN-gamma (Human) (SEQ ID NO: 76)ATGAAGTACACTAGCTATATATTGGCCTTCCAGCTTTGCATCGTATTGGGTAGCCTCGGATGCTATTGCCAAGACCCGTATGTCAAAGAAGCCGAAAATCTCAAAAAGTATTTCAATGCCGGACACTCAGACGTCGCGGATAACGGTACACTGTTTCTTGGCATCCTGAAAAATTGGAAGGAAGAGAGTGACAGAAAAATAATGCAGTCACAAATAGTGTCCTTTTACTTTAAGCTGTTCAAAAATTTCAAGGATGACCAAAGTATCCAGAAGAGTGTTGAAACTATCAAAGAGGACATGAATGTGAAATTCTTTAACAGTAATAAGAAGAAGCGCGATGACTTCGAGAAACTCACTAATTACAGCGTAACGGATCTTAACGTCCAACGCAAGGCAATCCACGAGCTTATACAGGTAATGGCTGAGCTTAGTCCCGCAGCCAAGACAGGGAAGAGAAAAAGGTCTCAAATGCTTTTTCGGGGCCGGCGAGCTTCA CAAtaGIFN-gamma (Mouse) (SEQ ID NO: 77)ATGAACGCTACGCATTGCATCCTCGCACTCCAATTGTTCCTCATGGCTGTGTCAGGGTGTTACTGTCACGGTACTGTCATAGAAAGCCTCGAATCCCTGAATAACTATTTTAACAGTAGCGGTATAGATGTAGAAGAAAAGTCTCTCTTTCTTGACATCTGGAGGAATTGGCAAAAGGATGGAGACATGAAGATTCTCCAATCTCAGATTATATCATTTTACTTGAGGCTTTTTGAGGTTCTGAAGGATAACCAGGCGATCAGCAATAATATCAGCGTAATTGAATCTCACCTTATTACAACATTTTTCTCAAATTCCAAGGCAAAGAAAGATGCTTTCATGTCTATCGCGAAATTTGAGGTGAACAATCCTCAGGTACAAAGGCAAGCCTTTAACGAGCTGATTAGAGTTGTACATCAGTTGTTGCCCGAAAGTAGTCTTAGAAAACGC AAACGGAGCCGATGCtaGIFN-alpha (Mouse) (SEQ ID NO: 78)ATGGCAAGGTTGTGCGCTTTTCTCATGGTACTGGCTGTGCTCTCCTATTGGCCTACTTGTTCTCTGGGATGCGACTTGCCACAGACCCACAATCTGCGGAATAAGAGGGCTCTGACTCTGCTGGTGCAAATGAGACGGCTCTCTCCACTTAGCTGTTTGAAAGATAGAAAGGATTTCGGGTTCCCCCAGGAGAAGGTGGATGCCCAGCAGATCAAGAAGGCACAGGCTATCCCCGTCCTTTCCGAGCTGACCCAGCAAATTTTGAACATCTTTACAAGTAAGGATAGTTCAGCTGCATGGAATACCACACTTTTGGATTCTTTTTGTAACGATCTGCATCAGCAGCTGAACGATCTCCAGGGATGCCTGATGCAGCAAGTCGGCGTGCAAGAATTTCCACTCACCCAGGAGGACGCTCTGCTCGCAGTGCGAAAGTATTTTCACCGAATTACCGTGTACCTCCGGGAGAAAAAGCATTCACCCTGCGCTTGGGAAGTAGTCAGGGCCGAAGTATGGAGAGCCCTTAGTAGCTCCGCTAATGTACTGGGCCGGTTGCGGGAAGAGAAAtaG CCL21 (Human) (SEQ ID NO: 79)ATGGCGCAAAGTCTGGCTCTTTCACTCCTGATCCTGGTCTTGGCCTTCGGGATTCCGAGGACCCAAGGAAGTGATGGTGGCGCCCAAGATTGTTGCCTTAAATACAGCCAGCGGAAAATACCCGCGAAAGTGGTCAGGAGTTATAGAAAACAGGAGCCTTCCCTGGGTTGTAGTATCCCCGCCATACTTTTCCTCCCGAGAAAACGGAGCCAGGCCGAACTGTGCGCTGACCCTAAGGAACTTTGGGTGCAACAACTTATGCAACACCTGGATAAGACACCTTCTCCTCAAAAGCCAGCTCAGGGCTGCCGAAAAGATAGAGGCGCCTCAAAAACCGGAAAAAAGGGCAAAGGTTCTAAAGGATGTAAGCGGACTGAACGCTCTCAAACGCCTAAAGGGCCGtaGCCL21a (Mouse) (SEQ ID NO: 80)ATGGCGCAAATGATGACCCTTTCCCTGCTGAGTCTTGTCCTCGCGCTCTGCATCCCGTGGACGCAGGGGTCTGATGGGGGGGGCCAAGACTGTTGCCTGAAGTATTCACAAAAAAAGATACCGTACTCTATTGTCAGAGGGTACAGGAAGCAAGAACCCTCCTTGGGTTGCCCTATACCAGCAATTCTTTTCTCCCCACGCAAGCATTCCAAACCAGAACTGTGTGCGAACCCCGAGGAGGGTTGGGTACAGAACTTGATGCGAAGGCTTGACCAGCCCCCAGCCCCTGGCAAGCAGTCACCTGGGTGCAGAAAAAACAGAGGTACTTCAAAGAGCGGCAAGAAAGGCAAAGGGAGTAAAGGATGTAAAAGAACGGAGCAGACCCAGCCTTCACGAGGCtaGTail-less CCL21 (Human) (SEQ ID NO: 81)ATGGCGCAAAGTCTGGCTCTTTCACTCCTGATCCTGGTCTTGGCCTTCGGGATTCCGAGGACCCAAGGAAGTGATGGTGGCGCCCAAGATTGTTGCCTTAAATACAGCCAGCGGAAAATACCCGCGAAAGTGGTCAGGAGTTATAGAAAACAGGAGCCTTCCCTGGGTTGTAGTATCCCCGCCATACTTTTCCTCCCGAGAAAACGGAGCCAGGCCGAACTGTGCGCTGACCCTAAGGAACTTTGGGTGCAACAACTTATGCAACACCTGGATAAGACACCTTCTCCTCAAAAGCCAGCTCAGGGCtaGTail-less CCL21 (Mouse) (SEQ ID NO: 82)ATGGCGCAAATGATGACCCTTTCCCTGCTGAGTCTTGTCCTCGCGCTCTGCATCCCGTGGACGCAGGGGTCTGATGGGGGGGGCCAAGACTGTTGCCTGAAGTATTCACAAAAAAAGATACCGTACTCTATTGTCAGAGGGTACAGGAAGCAAGAACCCTCCTTGGGTTGCCCTATACCAGCAATTCTTTTCTCCCCACGCAAGCATTCCAAACCAGAACTGTGTGCGAACCCCGAGGAGGGTTGGGTACAGAACTTGATGCGAAGGCTTGACCAGCCCCCAGCCCCTGGCAAGCAGTCACCTGGGtaG CCL19 (Mouse) (SEQ ID NO: 83)ATGGCACCCCGCGTCACACCCTTGCTTGCTTTTTCTCTGCTTGTCCTCTGGACCTTCCCCGCTCCTACCCTTGGAGGAGCCAATGATGCCGAGGATTGCTGCCTGAGTGTTACACAAAGGCCAATACCAGGGAATATAGTGAAGGCATTCCGGTATCTGCTCAATGAAGATGGGTGCAGAGTCCCCGCAGTTGTCTTTACAACATTGCGAGGTTACCAGCTTTGTGCTCCCCCAGACCAGCCTTGGGTAGATCGCATTATTCGCCGGTTGAAGAAGAGCTCAGCAAAGAATAAGGGCAATTCCACACGGAGAAGC CCCGTCTCCtaGCCL19 (Mouse) (SEQ ID NO: 84)ATGAAATCAGCAGTCCTTTTCTTGCTCGGGATTATTTTTCTGGAACAATGTGGAGTGAGGGGAACACTCGTAATAAGAAACGCTCGGTGCTCATGCATATCAACATCACGGGGCACTATCCACTACAAATCCCTGAAGGATCTGAAGCAGTTCGCCCCAAGCCCTAACTGTAACAAGACCGAAATTATCGCAACTCTCAAAAATGGAGATCAGACTTGTCTTGACCCAGATTCAGCAAATGTCAAGAAGCTGATGAAAGAGTGGGAAAAGAAGATTTCACAAAAAAAAAAGCAAAAACGCGGCAAGAAACATCAAAAGAACATGAAAAACAGGAAACCTAAGACTCCCCAGTCAAGGAGA AGATCCCGCAAGACAACCtaGCXCL11 (Mouse) (SEQ ID NO: 85)ATGAACAGAAAAGTTACCGCTATAGCACTTGCTGCCATAATATGGGCCACCGCAGCTCAAGGGTTCCTGATGTTCAAGCAGGGCCGATGCCTCTGCATTGGCCCTGGAATGAAGGCCGTGAAAATGGCCGAAATAGAAAAAGCTAGTGTCATATACCCCTCTAACGGTTGCGATAAAGTCGAGGTTATAGTCACAATGAAAGCTCATAAACGCCAACGCTGCCTCGACCCCCGGTCTAAGCAGGCTAGGCTCATAATGCAAGCAATCGAGAAGAAAAACTTTCTTAGACGGCAAAACATGtaG CXCL10 (Mouse) (SEQ ID NO: 86)ATGAACCCATCTGCCGCCGTTATTTTCTGTCTGATACTCCTTGGGCTGAGTGGCACACAAGGCATACCCCTCGCCCGCACAGTCCGGTGTAATTGTATACATATTGACGACGGCCCTGTTAGAATGCGGGCCATCGGTAAGCTGGAGATTATACCAGCAAGCCTTAGTTGTCCCAGGGTTGAAATCATAGCAACTATGAAAAAAAACGACGAACAAAGATGTTTGAATCCCGAGAGCAAGACAATCAAAAACCTTATGAAAGCATTTAGTCAAAAACGCTCTAAACGCGCTCCAtaG CXCL10 (Human) (SEQ ID NO: 87)ATGAATCAGACGGCAATCCTTATATGCTGCCTTATATTCCTTACTCTCTCAGGGATACAAGGGGTACCACTTTCTCGGACTGTTCGCTGCACTTGCATTTCAATATCTAACCAACCTGTAAATCCGCGGAGCCTGGAAAAATTGGAGATTATACCTGCTTCTCAATTCTGCCCTCGGGTGGAAATCATCGCCACTATGAAGAAGAAGGGCGAGAAAAGGTGTCTGAATCCAGAGTCAAAGGCAATCAAAAACCTGCTGAAAGCGGTGTCAAAGGAACGGTCCAAGAGATCACCCtaG CXCL11-CXCL 10 (Mouse) (SEQ ID NO: 88)ATGAACAGGAAAGTAACAGCCATTGCATTGGCTGCCATCATCTGGGCCACCGCAGCACAGGGTTTTCTGATGTTTAAGCAAGGGCGCTGTCTCTGTATAGGCCCAGGCATGAAGGCCGTGAAGATGGCAGAGATTGAGAAGGCATCTGTGATTTATCCTTCTAACGGGTGCGATAAAGTCGAAGTTATTGTGACAATGAAGGCACACAAACGCCAACGGTGTTTGGACCCACGATCTAAACAGGCAAGATTGATTATGCAAGCCATCGAGAAAAAGAACTTTCTCCGAAGGCAAAATATGATCCCTTTGGCTCGGACAGTGCGGTGTAACTGTATTCACATCGACGATGGGCCAGTACGGATGAGAGCAATAGGAAAGCTCGAAATCATACCCGCCTCATTGTCTTGTCCCAGGGTGGAAATAATCGCCACTATGAAAAAGAACGATGAACAGAGGTGTCTCAACCCAGAGAGTAAGACTATCAAGAACCTTATGAAGGCATTCAGTCAGAAGAGGTCAAAGCGAGCACCAtaG XCL 1 (Human) (SEQ ID NO: 89)ATGAGACTTCTCATATTGGCGCTTCTCGGGATATGTTCTCTTACGGCATACATAGTTGAGGGGGTGGGATCTGAGGTTAGCGATAAACGAACTTGTGTTAGTCTTACAACACAGAGGCTTCCAGTCTCCAGGATAAAAACATATACGATAACTGAGGGATCTCTCAGAGCGGTCATCTTCATAACGAAGAGGGGCCTGAAGGTCTGTGCTGACCCACAAGCGACTTGGGTAAGGGACGTTGTGCGGAGCATGGACAGGAAGAGCAATACTCGCAACAACATGATCCAAACCAAACCTACGGGCACCCAACAGTCAACCAATACTGCGGTAACATTGACGGGGtaG XCL 1 (Mouse) (SEQ ID NO: 90)ATGCGCCTCCTTCTGCTGACTTTTCTGGGTGTATGTTGCCTGACACCCTGGGTCGTAGAAGGAGTAGGAACCGAGGTTCTGGAAGAGTCCTCATGTGTAAACTTGCAGACACAACGACTCCCCGTCCAAAAAATCAAGACCTATATAATCTGGGAGGGGGCAATGCGGGCCGTCATTTTCGTGACTAAACGAGGTCTCAAAATCTGCGCCGACCCCGAGGCTAAGTGGGTGAAGGCAGCCATTAAGACCGTGGATGGGAGAGCCAGCACCAGAAAGAACATGGCCGAAACAGTACCTACTGGCGCACAGCGGTCAACCTCAACTGCTATAACCTTGACAGGAtaG m sCD4OL #1 (SEQ ID NO: 91)ATGGAGACTGACACTCTGCTTCTGTGGGTGTTGCTGCTGTGGGTGCCTGGCAGTACAGGCGATATGCAACGAGGTGACGAGGACCCTCAAATCGCCGCCCATGTAGTCTCTGAAGCTAATAGCAACGCTGCATCCGTCTTGCAGTGGGCAAAGAAAGGCTACTATACTATGAAGTCCAACTTGGTAATGCTTGAAAACGGCAAGCAGTTGACTGTCAAGAGAGAGGGACTTTATTACGTCTATACCCAAGTCACATTCTGTAGCAATCGAGAACCCTCCTCACAGAGGCCTTTTATAGTGGGACTCTGGCTTAAACCAAGTAGCGGCTCTGAGCGCATACTGTTGAAAGCCGCAAACACACACAGCTCTTCCCAACTCTGCGAGCAGCAATCCGTGCATCTCGGTGGAGTATTTGAGCTTCAAGCCGGTGCCTCAGTGTTTGTGAACGTCACTGAGGCCTCCCAGGTCATACATCGAGTTGGGTTCAGCTCCTTC GGCTTGCTCAAGCTCtaGm sCD40L #2 (SEQ ID NO: 92)ATGGAAACTGATACATTGCTGCTCTGGGTTTTGCTGCTCTGGGTGCCTGGGAGTACAGGCGACATGAGGAGGCAGTTCGAGGATCTCGTTAAGGATATTACCCTTAATAAGGAGGAGAAGAAAGAAAACTCTTTTGAGATGCAACGAGGGGACGAAGATCCTCAGATCGCTGCTCACGTGGTCTCTGAAGCTAACAGCAACGCCGCTTCTGTCCTCCAGTGGGCCAAGAAAGGTTATTACACCATGAAATCAAACCTTGTAATGCTTGAAAACGGGAAACAGCTTACAGTGAAGAGGGAAGGTCTTTACTACGTCTATACCCAGGTAACCTTCTGCTCAAACAGAGAACCATCAAGCCAGAGGCCATTCATAGTGGGGCTCTGGCTCAAACCTTCCAGTGGCAGCGAGAGAATCTTGTTGAAAGCTGCTAATACACATAGTAGTAGCCAGCTTTGCGAGCAACAGTCAGTCCACCTCGGGGGGGTGTTTGAGTTGCAAGCAGGGGCCTCAGTATTCGTGAATGTCACTGAGGCTTCCCAGGTAATTCACAGGGTAGGCTTTAGTTCATTCGGTTTGCTGAAGCTT taGm sCD40L #3 (SEQ ID NO: 93)ATGCGAAGAATGCAGCTTCTGCTCCTTATTGCTCTGAGTCTCGCCCTTGTCACCAACTCCGGGGACAGAATGAAACAAATCGAGGACAAAATTGAAGAAATACTGAGTAAAATATATCACATCGAAAACGAAATTGCACGCATTAAGAAATTGATTGGCGAACGCACCAGTGGCGGCTCTGGTGGCACCGGAGGTTCAGGCGGGACCGGGGGCTCTGACAAAGTCGAAGAGGAGGTTAACCTTCATGAGGACTTTGTGTTCATCAAGAAGCTGAAACGGTGCAATAAAGGAGAAGGTTCTTTGAGCCTCCTTAATTGCGAAGAGATGCGACGACAGTTCGAGGATCTGGTTAAGGACATTACACTTAATAAGGAAGAGAAAAAGGAGAACTCTTTCGAAATGCAGCGCGGCGATGAAGATCCCCAGATAGCCGCCCATGTCGTCTCTGAGGCCAACTCTAACGCAGCATCCGTCCTCCAGTGGGCTAAGAAAGGATATTATACTATGAAAAGCAATTTGGTCATGCTCGAAAACGGTAAACAGCTCACTGTTAAGAGAGAAGGCCTCTATTACGTATATACTCAAGTAACTTTCTGTTCTAATAGGGAACCCTCCTCTCAAAGACCTTTTATCGTAGGACTCTGGTTGAAACCAAGTAGCGGTAGTGAAAGGATTCTGCTCAAAGCAGCTAATACTCACTCCAGCAGTCAACTGTGCGAACAACAAAGCGTTCACCTCGGGGGCGTCTTTGAACTTCAGGCAGGTGCCAGTGTTTTCGTCAACGTAACAGAAGCATCCCAGGTAATTCATCGAGTAGGGTTTTCTAGCTTTGGITTGCTGAAGCTGtaG anti-CD40_FGK4.5 (SEQ ID NO: 94)ATGGAAACTGATCGCCTGTTGCTCTGGGTACTTCTTCTGTGGGTGCCTGGGTCCACTGGTGACACTGTACTTACACAATCACCCGCTTTGGCCGTTTCTCCTGGTGAACGGGTCACAATTAGTTGCCGAGCTTCCGATTCTGTATCTACTCTTATGCATTGGTATCAACAAAAACCTGGTCAGCAGCCAAAATTGCTCATTTATCTTGCTAGTCACTTGGAGTCCGGCGTACCTGCTCGATTCAGCGGTAGTGGGTCTGGCACAGATTTCACTTTGACCATAGATCCCGTGGAGGCCGATGACACTGCAACCTACTATTGCCAGCAATCCTGGAACGACCCTTGGACTTTCGGCGGCGGCACCAAGCTGGAACTCAAGCGAGCAGATGCTGCCCCAACCGTTAGTATATTCCCACCCTCAACCGAACAACTCGCCACAGGAGGCGCTAGTGTCGTGTGTCTTATGAACAATTTCTATCCACGAGACATTAGCGTCAAGTGGAAAATTGATGGGACAGAAAGGCGAGATGGAGTTTTGGATTCAGTAACAGACCAGGATTCAAAGGATTCTACCTATAGCATGAGCTCCACCTTGAGCCTGACCAAAGCTGATTATGAATCTCATAACCTGTATACTTGTGAAGTGGTGCATAAGACTTCTAGCTCACCAGTGGTTAAATCTTTTAACCGCAACGAATGTCGGCGCAAGAGGGGTTCCGGAGAGGGAAGGGGTAGTCTGCTCACCTGCGGCGATGTTGAAGAAAATCCTGGTCCCATGGACATTCGGCTCTCTTTGGTATTCCTGGTACTTTTTATAAAGGGGGTGCAATGTGAAGTCCAGCTCGTGGAAAGCGGTGGGGGCCTGGTTCAGCCCGGTCGCAGCCTTAAACTTAGTTGCGCAGCATCCGGATTTACATTTTCTGACTATAACATGGCCTGGGTTCGACAGGCACCCAAAAAAGGGCTGGAGTGGGTCGCAACTATCATATACGATGGTTCCCGGACATACTATAGAGATTCAGTGAAGGGGCGCTTTACAATAAGCAGGGACAATGCTAAGTCTACCTTGTATCTTCAGATGGACTCCCTGAGGAGCGAAGATACAGCAACATATTATTGTGCTACAAACCGCTGGTTGCTGCTTCATTATTTCGACTACTGGGGTCAGGGCGTCATGGTAACTGTATCAAGCGCCGAGACCACAGCCCCTTCTGTATATCCATTGGCACCAGGTACTGCTCTGAAATCCAACTCAATGGTAACCCTTGGATGTCTGGTTAAGGGTTATTTTCCCGAGCCCGTCACAGTTACTTGGAACTCTGGGGCCCTTTCTAGCGGAGTCCATACCTTTCCCGCCGTTTTGCAGAGTGGTCTGTACACCCTTACCTCAAGCGTCACAGTTCCATCTAGCACATGGAGCTCCCAGGCAGTAACTTGTAATGTGGCCCATCCAGCCTCCTCAACTAAGGTAGATAAAAAGATCGTTCCCAGAGAATGCAATCCATGTGGATGCACCGGGTCTGAGGTCAGCAGTGTGTTCATTTTCCCACCCAAGACTAAAGATGTATTGACTATTACTCTTACACCCAAAGTAACCTGCGTGGTGGTTGATATTAGTCAAAATGATCCCGAGGTACGGTTCTCTTGGTTTATCGACGACGTCGAAGTACATACAGCTCAGACACACGCTCCCGAGAAACAAAGCAATTCCACTCTTAGGAGCGTGTCCGAGTTGCCAATCGTACATAGGGATTGGCTTAATGGCAAGACCTTTAAGTGTAAGGTCAATTCAGGGGCATTCCCCGCACCAATAGAGAAGAGTATAAGCAAACCCGAGGGGACACCCAGAGGTCCACAGGTCTATACAATGGCTCCCCCCAAGGAAGAGATGACCCAAAGTCAAGTCTCAATTACATGTATGGTGAAGGGCTTTTATCCACCCGACATATACACTGAGTGGAAGATGAATGGACAGCCCCAAGAGAATTATAAAAACACTCCCCCTACCATGGACACCGACGGGTCCTATTTTCTTTATAGTAAATTGAACGTGAAAAAGGAGACCTGGCAACAAGGCAACACTTTCACCTGCTCCGTTCTTCACGAGGGCCTGCATAATCATCATACCGAAAAGTCTCTCAGTCATTCTCCAGGTAAGtaGCD40L 2 (Human) (SEQ ID NO: 95)ATGGAAACAGATACGTTGCTGTTGTGGGTACTTCTCCTTTGGGTCCCTGGCAGCACAGGGGACGAGAATAGTTTCGAAATGCAGAAGGGCGACCAGAACCCACAGATCGCGGCTCACGTTATATCAGAAGCAAGTAGTAAGACCACTTCCGTACTTCAGTGGGCTGAAAAAGGATATTACACCATGTCCAACAATCTCGTGACACTGGAGAACGGTAAACAACTTACGGTGAAACGACAGGGCCTCTATTACATCTACGCTCAGGTGACATTCTGCTCAAATAGGGAGGCTTCTAGTCAAGCGCCCTTCATCGCCAGCCTGTGCCTCAAATCTCCCGGCCGGTTCGAACGAATCCTGTTGCGAGCGGCCAATACCCATAGCTCAGCTAAACCTTGCGGCCAGCAGAGTATTCATCTTGGTGGTGTGTTTGAACTTCAGCCGGGAGCATCTGTGTTCGTCAACGTAACGGACCCTAGCCAAGTGTCTCATGGGACAGGTTTTACATCCTTCGGACTCCTCAAGTTGtaG Flt3L (Human) (SEQ ID NO: 96)ATGACAGTTCTCGCGCCAGCTTGGAGTCCCACCACATACTTGCTTTTGCTTCTGCTTCTGTCCTCTGGCCTGAGTGGGACCCAAGATTGTTCCTTTCAACATTCCCCAATTAGTTCTGATTTTGCAGTGAAGATTAGAGAGCTCTCAGACTATCTGCTGCAAGATTATCCTGTCACAGTCGCTTCAAACCTGCAAGACGAAGAGCTCTGCGGTGCCTTGTGGCGGTTGGTCTTGGCTCAAAGATGGATGGAGAGACTGAAAACCGTAGCAGGCAGCAAGATGCAGGGTCTCCTGGAAAGGGTGAACACGGAAATCCATTTTGTGACCAAGTGCGCGTTCCAGCCCCCACCGAGTTGTCTCCGGTTTGTTCAAACGAATATATCCCGGTTGCTCCAGGAAACCTCAGAACAACTGGTGGCTTTGAAACCCTGGATCACAAGACAAAACTTTAGTCGGTGCCTCGAACTCCAGTGCCAACCAGATTCTTCTACACTTCCCCCCCCGTGGTCCCCGCGCCCGTTGGAAGCAACGGCCCCAtaGTGFb TRAP (Human) (SEQ ID NO: 97)ATGGCCTGGAGTCCTCTGTTTCTGACTCTTATAACTCACTGTGCCGGCAGTTGGGCTATACCCCCTCATGTACAGAAGTCTGTAAACAACGACATGATTGTAACCGACAATAATGGCGCAGTGAAATTCCCACAACTGTGTAAGTTCTGTGATGTACGGTTTAGTACATGCGACAATCAAAAAAGCTGTATGTCTAACTGCTCTATTACATCCATATGTGAAAAACCTCAGGAGGTGTGTGTTGCCGTTTGGCGAAAAAATGATGAGAATATCACACTGGAGACAGTATGTCATGACCCTAAACTGCCATACCATGATTTCATACTGGAGGACGCCGCCAGTCCTAAGTGCATTATGAAAGAGAAAAAGAAACCCGGTGAAACATTCTTTATGTGCTCTTGTAGCTCTGACGAGTGTAACGACAACATTATATTCAGCGAGGAGTACAATACAAGCAACCCCGATATACCACCTCACGTACAAAAAAGTGTCAACAACGATATGATTGTTACCGACAATAACGGAGCTGTTAAGTTTCCTCAGTTGTGCAAGTTCTGCGATGTACGATTCTCTACCTGCGACAACCAAAAGTCATGTATGTCTAACTGTTCCATAACCTCCATCTGCGAGAAGCCCCAGGAAGTCTGCGTCGCCGTGTGGCGGAAAAACGACGAGAATATCACTCTTGAAACCGTTTGTCATGATCCTAAACTGCCCTATCACGACTTTATTCTGGAAGATGCTGCTTCCCCTAAGTGTATCATGAAAGAAAAGAAGAAACCTGGGGAGACATTCTTTATGTGTTCATGCTCCTCCGATGAGTGTAACGACAATATCATCTTCTCTGAGGAATAC AACACTTCTAACCCTGATtaGFresolimumab (Human) (SEQ ID NO: 98)ATGGCCTGGTCCCCTCTTTTTCTGACCCTCATCACACACTGTGCAGGCTCATGGGCTGAGACCGTCTTGACCCAGTCCCCAGGAACTTTGTCTCTGTCTCCTGGTGAAAGAGCTACCCTTAGTTGTCGAGCCTCTCAGTCCCTTGGTTCTAGCTATCTCGCTTGGTACCAGCAAAAGCCAGGCCAGGCCCCACGACTGCTGATCTACGGAGCATCTTCACGGGCTCCCGGCATTCCCGATCGATTTTCCGGATCTGGTAGTGGTACAGATTTCACACTGACCATATCTCGCCTGGAGCCCGAGGACTTTGCTGTTTATTATTGTCAGCAGTACGCCGATTCTCCTATCACTTTTGGACAGGGAACCCGCCTGGAGATTAAGCGCACAGTAGCAGCTCCATCCGTCTTTATCTTTCCACCATCAGATGAACAGCTCAAGAGTGGGACCGCAAGTGTAGTATGCCTGCTGAACAATTTTTACCCTAGAGAGGCCAAAGTGCAGTGGAAGGTGGATAACGCCCTCCAGAGTGGCAATAGTCAAGAAAGTGTTACTGAGCAAGATAGTAAGGACTCTACATACTCTTTGAGTTCTACTTTGACCCTGTCAAAAGCAGATTATGAAAAACATAAGGTGTATGCATGTGAAGTTACACACCAAGGGTTGTCCTCTCCAGTTACAAAATCTTTTAATAGAGGAGAGTGCCGCCGCAAACGCGGTAGTGGAGAAGGTCGAGGCTCACTCTTGACCTGTGGCGACGTGGAAGAAAATCCCGGTCCTATGGATTGGACTTGGAGGGTATTTTGTCTTTTGGCAGTAACACCTGGAGCTCACCCCCAAGTACAGCTCGTCCAATCTGGTGCCGAGGTTAAAAAGCCTGGAAGTTCAGTGAAGGTCTCTTGCAAGGCATCTGGATACACCTTTTCATCTAACGTCATATCCTGGGTACGGCAAGCCCCAGGACAGGGACTTGAGTGGATGGGAGGGGTCATCCCCATCGTGGACATTGCTAATTACGCTCAGCGATTCAAAGGGCGGGTTACTATAACTGCCGACGAGTCTACCTCAACTACCTACATGGAGTTGTCCTCTCTCCGCTCCGAGGACACTGCTGTATATTACTGTGCCAGCACTCTCGGGTTGGTGTTGGATGCCATGGACTATTGGGGACAAGGAACCCTGGTGACAGTTAGCTCCGCAAGCACTAAAGGCCCTTCTGTTTTTCCCTTGGCACCTTGTAGTAGGTCTACCTCTGAGTCTACAGCAGCACTTGGATGCTTGGTTAAGGACTATTTTCCCGAGCCAGTTACAGTCTCTTGGAACAGTGGTGCCCTCACAAGTGGGGTTCATACCTTCCCCGCAGTCCTCCAGAGTAGTGGCCTTTACAGCCTCTCATCAGTTGTGACTGTTCCTAGTTCATCACTCGGTACTAAGACATATACATGTAACGTAGACCACAAGCCAAGCAACACAAAAGTAGACAAACGAGTCGAATCTAAGTATGGACCCCCTTGTCCCTCCTGTCCTGCTCCCGAGTTCCTTGGGGGCCCTTCCGTGTTCTTGTTTCCTCCCAAGCCCAAGGATACCCTCATGATCTCACGAACCCCAGAGGTAACATGTGTGGTTGTTGACGTAAGTCAGGAAGATCCCGAAGTGCAATTTAATTGGTACGTGGATGGCGTCGAAGTCCATAACGCTAAAACAAAACCCCGAGAGGAACAATTCAATTCCACATATCGGGTGGTGAGTGTATTGACCGTTCTTCACCAAGATTGGCTGAACGGCAAGGAGTATAAGTGTAAAGTAAGCAACAAAGGTCTGCCAAGTAGCATAGAAAAAACAATATCTAAAGCTAAGGGCCAACCAAGGGAACCACAAGTATATACATTGCCCCCCTCTCAGGAAGAGATGACAAAGAATCAAGTTAGCCTGACCTGTTTGGTAAAGGGGTTCTATCCCTCAGATATAGCAGTCGAGTGGGAATCTAACGGCCAGCCCGAGAATAATTATAAAACAACCCCCCCTGTGTTGGACTCAGACGGCAGCTTCTTTCTCTATTCACGGCTCACTGTTGATAAGTCCCGATGGCAGGAGGGGAATGTTTTCAGCTGTAGCGTGATGCACGAAGCTCTCCACAACCACTATACACAGAAAAGTTTGTCT TTGTCCCTTGGAAAAtaGTGFb neutralizing peptide (Human) (SEQ ID NO: 99)ATGAGTACATCCTTTCCAGAGCTGGATCTGGAGAATTTTGAGTATGACGACAGTGCCGAAGCCTGCTACCTCGGGGACATAGTCGCATTCGGGACAATCTTTTTGTCTGTATTTTACGCCCTGGTGTTTACATTTGGCCTGGTTGGAAATCTGTTGGTCGTACTCGCTCTCACCAATTCCCGAAAACCCAAAAGTATAACAGACATATACCTGTTGAATCTGGCACTGAGTGACCTTTTGTTCGTCGCCACCCTTCCTTTTTGGACACACTACCTTATCAGTCACGAGGGGCTTCATAATGCTATGTGCAAGCTCACTACTGCCTTCTTCTTTATCGGATTCTTCGGGGGTATCTTTTTTATCACAGTTATTAGCATTGACCGATACCTTGCCATAGTGCTCGCAGCCAACTCAATGAACAACCGCACCGTGCAGCATGGAGTGACTATTTCCTTGGGTGTGTGGGCCGCTGCTATACTTGTCGCCAGCCCTCAATTCATGTTTACCAAAAGGAAAGACAATGAGTGCCTCGGAGATTACCCTGAGGTGTTGCAAGAAATGTGGCCTGTACTTCGAAATAGCGAAGTGAATATACTCGGCTTTGCTCTTCCTCTGCTCATCATGTCATTCTGTTATTTTCGAATAATCCAAACATTGTTCAGCTGTAAGAACCGAAAGAAAGCCCGCGCCGTACGCCTGATTCTGCTCGTTGTGTTCGCCTTTTTTCTGTTTTGGACTCCTTACAACATAATGATATTCCTGGAGACTCTCAAATTCTATAACTTTTTTCCCTCCTGTGATATGAAAAGGGACCTTAGATTGGCTCTCAGTGTCACTGAAACAGTAGCCTTTAGCCATTGTTGTCTCAACCCTTTCATATATGCATTTGCAGGGGAAAAGTTCCGGCGGTATCTCGGACATTTGTATCGGAAGTGCTTGGCCGTGTTGTGTGGTCATCCTGTCCATACCGGATTCTCTCCTGAGAGTCAACGGAGCCGCCAAGATTCAATCCTGTCCAGTTTCACTCACTATACTTCAGAGGGGGATGGCAGCCTTCTGCTC Kynureinase #1 (SEQ ID NO: 100)ATGGAGACCGACACTTTGTTGCTGTGGGTACTTTTGTTGTGGGTCCCAGGATCTACCGGGGATATGGAACCCTCTCCTCTTGAACTGCCAGTAGACGCCGTGCGCCGCATTGCAGCCGAGTTGAATTGCGATCCAACAGATGAACGCGTTGCCCTGAGGCTCGACGAAGAGGATAAATTGTCACATTTCAGGAACTGCTTTTACATTCCAAAGATGAGGGATCTTCCATCCATAGATCTTAGCCTCGTGTCCGAGGATGACGATGCCATATATTTTCTTGGGAACAGTCTTGGGTTGCAGCCAAAAATGGTACGGACATATCTCGAAGAGGAGCTGGACAAATGGGCTAAAATGGGTGCTTACGGCCACGACGTGGGAAAACGCCCCTGGATAGTTGGCGACGAATCTATCGTGAGTCTTATGAAAGATATAGTTGGAGCACATGAGAAAGAAATTGCACTGATGAATGCCCTTACTATCAATCTGCATCTCCTCTTGCTTTCATTCTTTAAGCCCACTCCTAAACGCCACAAAATACTTTTGGAAGCAAAAGCCTTTCCAAGCGACCACTACGCTATTGAGTCACAAATACAACTCCATGGACTTGATGTGGAAAAGTCTATGCGGATGGTAAAACCACGCGAAGGCGAGGAGACCCTTCGAATGGAGGACATACTTGAGGTCATCGAAGAAGAAGGAGATAGTATAGCAGTTATCCTTTTCAGCGGGCTGCACTTCTACACAGGTCAACTCTTTAACATTCCAGCTATTACTAAGGCAGGCCACGCTAAAGGATGCTTCGTGGGCTTTGACCTTGCACACGCAGTAGGAAACGTAGAGCTCCGCTTGCACGATTGGGGCGTTGATTTCGCCTGCTGGTGTTCATATAAGTATCTTAACTCAGGAGCTGGTGGGTTGGCAGGCGCATTCGTACACGAGAAACACGCTCATACCGTAAAGCCTGCACTGGTAGGGTGGTTCGGACACGATCTCTCTACCCGCTTCAATATGGATAATAAACTCCAGCTTATACCTGGCGCCAATGGATTCAGGATCTCAAATCCTCCTATTTTGCTCGTTTGCAGTTTGCACGCATCTCTTGAGGTGTTCCAGCAGGCTACCATGACTGCACTCCGCCGGAAGTCAATCCTTTTGACCGGATACTTGGAGTATATGCTGAAACATTATCACTCAAAAGATAACACTGAGAATAAGGGCCCCATAGTAAACATTATCACTCCATCTCGGGCTGAAGAGCGCGGCTGCCAACTCACATTGACTTTTTCCATTCCCAAGAAGTCAGTGTTCAAAGAGTTGGAGAAACGGGGGGTTGTATGTGATAAGCGGGAGCCAGATGGAATCCGCGTTGCCCCAGTCCCCCTCTATAATTCTTTTCACGATGTATACAAGTTTATTAGACTGCTGACAAGTATCTTGGAC TCATCTGAGCGATCTtaGKynureinase #2 (SEQ ID NO: 101)ATGGAACCCTCTCCTCTTGAACTGCCAGTAGACGCCGTGCGCCGCATTGCAGCCGAGTTGAATTGCGATCCAACAGATGAACGCGTTGCCCTGAGGCTCGACGAAGAGGATAAATTGTCACATTTCAGGAACTGCTTTTACATTCCAAAGATGAGGGATCTTCCATCCATAGATCTTAGCCTCGTGTCCGAGGATGACGATGCCATATATTTTCTTGGGAACAGTCTTGGGTTGCAGCCAAAAATGGTACGGACATATCTCGAAGAGGAGCTGGACAAATGGGCTAAAATGGGTGCTTACGGCCACGACGTGGGAAAACGCCCCTGGATAGTTGGCGACGAATCTATCGTGAGTCTTATGAAAGATATAGTTGGAGCACATGAGAAAGAAATTGCACTGATGAATGCCCTTACTATCAATCTGCATCTCCTCTTGCTTTCATTCTTTAAGCCCACTCCTAAACGCCACAAAATACTTTTGGAAGCAAAAGCCTTTCCAAGCGACCACTACGCTATTGAGTCACAAATACAACTCCATGGACTTGATGTGGAAAAGTCTATGCGGATGGTAAAACCACGCGAAGGCGAGGAGACCCTTCGAATGGAGGACATACTTGAGGTCATCGAAGAAGAAGGAGATAGTATAGCAGTTATCCTTTTCAGCGGGCTGCACTTCTACACAGGTCAACTCTTTAACATTCCAGCTATTACTAAGGCAGGCCACGCTAAAGGATGCTTCGTGGGCTTTGACCTTGCACACGCAGTAGGAAACGTAGAGCTCCGCTTGCACGATTGGGGCGTTGATTTCGCCTGCTGGTGTTCATATAAGTATCTTAACTCAGGAGCTGGTGGGTTGGCAGGCGCATTCGTACACGAGAAACACGCTCATACCGTAAAGCCTGCACTGGTAGGGTGGTTCGGACACGATCTCTCTACCCGCTTCAATATGGATAATAAACTCCAGCTTATACCTGGCGCCAATGGATTCAGGATCTCAAATCCTCCTATTTTGCTCGTTTGCAGTTTGCACGCATCTCTTGAGGTGTTCCAGCAGGCTACCATGACTGCACTCCGCCGGAAGTCAATCCTTTTGACCGGATACTTGGAGTATATGCTGAAACATTATCACTCAAAAGATAACACTGAGAATAAGGGCCCCATAGTAAACATTATCACTCCATCTCGGGCTGAAGAGCGCGGCTGCCAACTCACATTGACTTTTTCCATTCCCAAGAAGTCAGTGTTCAAAGAGTTGGAGAAACGGGGGGTTGTATGTGATAAGCGGGAGCCAGATGGAATCCGCGTTGCCCCAGTCCCCCTCTATAATTCTTTTCACGATGTATACAAGTTTATTAGACTGCTGACAAGTATCTTGGACTCATCTGAGCGATCTtaG VEGF (SEQ ID NO: 102)ATGAATTTCTTGCTGAGCTGGGTGCATTGGACACTCGCATTGTTGCTGTACTTGCACCATGCCAAGTGGTCCCAGGCTGCACCCACTACTGAGGGCGAGCAAAAGTCTCATGAGGTGATTAAATTTATGGACGTTTACCAACGATCATACTGTCGGCCAATCGAAACCCTCGTAGATATATTCCAGGAGTACCCAGACGAGATCGAATACATTTTCAAGCCCTCATGTGTCCCATTGATGCGATGTGCTGGGTGCTGTAACGACGAAGCACTTGAATGTGTCCCCACCTCCGAGAGTAACATCACAATGCAAATAATGAGAATCAAGCCCCACCAATCCCAACATATCGGTGAAATGTCATTCCTTCAGCATTCCCGCTGCGAGTGCCGGCCTAAGAAGGACCGCACCAAACCAGAGAACCATTGTGAACCCTGTTCTGAGAGACGGAAGCACTTGTTCGTACAGGACCCTCAAACATGCAAGTGCAGCTGTAAGAATACCGACTCACGGTGTAAAGCTAGGCAACTGGAGCTTAATGAAAGGACCTGCCGATGCGATAAACCCAGGAGGtaa GM-CSF (SEQ ID NO: 103)ATGTGGTTGCAGAATTTGCTCTTCCTGGGGATTGTGGTCTACAGCCTCTCCGCACCTACCCGCTCTCCTATCACAGTTACAAGACCCTGGAAACATGTGGAGGCCATTAAAGAAGCATTGAATTTGTTGGACGATATGCCCGTCACCCTGAATGAAGAAGTAGAAGTTGTTTCTAATGAGTTCAGCTTTAAAAAATTGACCTGTGTGCAGACACGGCTTAAAATTTTTGAACAGGGACTTAGAGGAAACTTTACTAAGCTGAAGGGGGCACTTAACATGACAGCTTCTTATTATCAGACCTATTGTCCTCCAACACCTGAAACCGACTGTGAAACACAGGTAACCACTTACGCCGATTTTATTGATTCTTTGAAAACATTCCTCACCGATATACCATTTGAGTGT AAGAAGCCAGGCCAAAAGtaGAnti-PD1 (SEQ ID NO: 104) ATGGAAACTGACACACTTCTTCTGTGGGTCTTGCTCCTGTGGGTCCCAGGCTCTACTGGTGACAGTCCTGATAGGCCATGGAACCCACCTACCTTTAGTCCAGCCTTGCTCGTCGTAACCGAAGGGGACAACGCTACATTCACCTGCTCTTTTAGCAATACTTCTGAGAGTTTTCATGTAGTCTGGCATCGGGAGAGTCCATCCGGACAAACAGATACTTTGGCCGCTTTTCCAGAGGATAGGTCTCAACCTGGGCAAGACGCAAGGTTTCGAGTCACACAGCTTCCTAACGGGAGAGATTTTCACATGTCTGTAGTTCGGGCACGCCGAAATGATTCTGGCACATATGTTTGCGGTGTGATCTCACTTGCTCCAAAGATTCAAATAAAGGAGAGCCTTCGCGCCGAGTTGCGGGTGACTGAGCGGGAGCCCAAGTCCTGCGACAAAACCCATACTTGTCCACCCTGTGGCGGCGGGTCATCCGGTGGCGGGTCTGGGGGGCAACCAAGAGAGCCACAGGTATATACTCTTCCCCCCAGCAGAGAAGAAATGACAAAAAACCAAGTGTCCCTGACATGTCTGGTTAAAGGATTTTATCCCAGTGACATTGCTGTAGAATGGGAATCCAATGGTCAACCCGAGAATAACTACAAAACCACTCCTCCAGTATTGGACAGTGACGGTTCCTTCTTCCTCTATTCCAAACTTACAGTGGATAAATCCCGCTGGCAGCAAGGGAATGTATTCAGCTGTAGTGTCATGCACGAAGCTCTTCATAACCATTATACACAGAAATCTCTTTCCCTGAGC CCAGGTAAAtaGAdenosine Deaminase (ADA) #1 (Mouse) (SEQ ID NO: 105)ATGGAGACTGATACACTTTTGCTCTGGGTTTTGCTCTTGTGGGTACCAGGGTCTACTGGAGATGCACAAACTCCTGCATTCAACAAGCCTAAGGTAGAGCTTCATGTCCATTTGGACGGAGCCATAAAACCTGAAACCATACTCTATTTCGGCAAGAAACGGGGTATAGCACTTCCCGCTGATACCGTGGAAGAGTTGAGAAATATCATTGGCATGGACAAACCTCTTAGCCTGCCTGGCTTTCTTGCAAAGTTCGACTACTATATGCCAGTTATAGCAGGGTGTAGAGAAGCAATAAAGCGAATCGCCTATGAGTTCGTTGAGATGAAGGCTAAAGAAGGAGTTGTTTACGTGGAAGTCCGGTACTCACCTCATCTGCTTGCTAATAGCAAGGTGGACCCAATGCCATGGAATCAAACTGAAGGTGATGTAACCCCTGACGATGTGGTCGATTTGGTCAATCAAGGTCTCCAAGAAGGCGAGCAGGCTTTCGGCATTAAGGTAAGAAGTATATTGTGCTGTATGCGACATCAACCTTCATGGTCCCTGGAGGTCCTCGAATTGTGCAAAAAGTACAATCAAAAAACAGTGGTCGCAATGGATCTCGCTGGAGATGAGACCATAGAAGGTTCCTCTCTTTTCCCCGGTCATGTCGAAGCATATGAAGGGGCTGTCAAAAATGGTATCCACCGCACCGTCCACGCAGGGGAAGTAGGGTCCCCAGAAGTAGTCAGGGAAGCCGTTGACATTTTGAAAACAGAAAGAGTCGGGCATGGCTACCATACAATAGAGGACGAAGCCTTGTACAATCGACTTTTGAAAGAAAATATGCACTTCGAGGTCTGTCCCTGGAGTTCATATCTCACCGGAGCATGGGACCCCAAAACAACCCACGCCGTCGTACGCTTCAAGAATGATAAGGCAAACTACAGTTTGAATACAGATGATCCACTGATATTCAAGTCAACACTTGACACTGACTACCAGATGACAAAAAAAGATATGGGTTTCACCGAAGAAGAGTTCAAGAGATTGAACATTAACGCAGCAAAAAGCTCCTTCCTGCCAGAGGAAGAGAAAAAAGAATTGCTTGAAAGGTT GTATCGAGAATACCAAAdenosine Deaminase (ADA) #2 (Mouse) (SEQ ID NO: 106)ATGGCACAAACTCCAGCTTTTAATAAGCCCAAAGTGGAACTTCATGTTCATCTGGATGGGGCAATTAAGCCCGAAACTATATTGTACTTTGGCAAAAAGAGGGGTATTGCCCTGCCAGCAGATACCGTTGAGGAGCTTCGCAACATCATTGGGATGGACAAGCCCCTCTCTCTGCCAGGTTTTCTCGCTAAATTCGATTATTATATGCCTGTTATTGCTGGTTGCCGGGAGGCCATCAAGAGGATAGCCTACGAGTTTGTTGAGATGAAGGCCAAAGAGGGCGTGGTGTACGTAGAGGTCAGATACAGCCCTCACCTGCTTGCCAACAGCAAGGTGGACCCAATGCCCTGGAACCAAACCGAGGGGGATGTCACTCCCGACGACGTTGTAGACCTCGTAAATCAGGGCCTTCAAGAGGGCGAGCAGGCATTTGGCATAAAAGTCCGGTCTATACTCTGCTGTATGAGGCACCAACCCTCCTGGTCTTTGGAGGTACTTGAGTTGTGTAAGAAATACAATCAAAAGACTGTAGTCGCCATGGATCTTGCAGGCGATGAAACCATCGAGGGTAGCTCCTTGTTCCCTGGACATGTTGAAGCCTACGAGGGGGCCGTAAAAAATGGGATACACAGGACTGTCCACGCTGGTGAAGTCGGAAGCCCAGAGGTGGTAAGGGAGGCAGTTGACATACTCAAGACAGAGCGGGTTGGACACGGATACCACACAATTGAGGACGAGGCCCTGTATAACCGCCTCCTCAAAGAGAACATGCATTTTGAGGTGTGTCCTTGGTCCAGCTACCTGACTGGTGCTTGGGACCCTAAAACAACTCACGCCGTGGTCCGGTTCAAGAACGATAAAGCCAATTACTCTTTGAATACCGACGACCCCCTCATATTCAAATCAACATTGGATACCGACTACCAAATGACCAAAAAGGATATGGGGTTTACTGAAGAGGAGTTCAAGAGGCTCAACATAAATGCCGCTAAATCCTCCTTTCTCCCCGAGGAAGAAAAAAAAGAACTCCTTGAGCGGCTGTATAGGGAGTATCAA4-1BBL #1 (Mouse) (SEQ ID NO: 107)ATGGAAACAGATACACTCTTGCTCTGGGTACTGCTTCTGTGGGTCCCCGGCTCTACTGGGGATGAAGATGATGTAACTACTACAGAAGAACTCGCTCCCGCTCTTGTCCCCCCACCCAAGGGTACCTGCGCCGGTTGGATGGCTGGCATCCCAGGACATCCAGGTCACAACGGTACCCCCGGAAGAGATGGTCGGGATGGAACTCCCGGCGAGAAGGGCGAAAAAGGGGATGCAGGGCTTCTGGGACCTAAAGGTGAAACAGGGGACGTTGGAATGACTGGTGCAGAAGGGCCTCGCGGCTTTCCTGGCACCCCTGGGAGGAAAGGAGAGCCCGGAGAGCTCCAGAGAACTGAACCTCGGCCTGCACTCACTATAACTACTTCCCCTAATCTTGGGACCCGCGAGAACAACGCCGATCAGGTTACACCTGTAAGCCATATCGGGTGCCCCAATACTACCCAGCAAGGGAGTCCCGTGTTCGCAAAGCTTTTGGCTAAAAACCAAGCATCCCTGTGTAACACTACTCTTAATTGGCATTCACAAGACGGTGCTGGTAGCTCTTATCTTTCTCAGGGGCTGCGGTACGAAGAAGATAAGAAGGAATTGGTTGTGGATTCTCCAGGACTCTATTATGTCTTTCTCGAATTGAAGCTCAGTCCCACCTTCACAAACACTGGACACAAAGTCCAGGGCTGGGTAAGTCTGGTACTCCAAGCAAAGCCCCAGGTTGACGATTTCGACAATTTGGCACTCACCGTAGAGCTTTTCCCATGCTCCATGGAAAATAAACTTGTTGATCGGTCATGGTCACAGCTCTTGCTGCTTAAGGCAGGGCATCGCCTCTCAGTGGGTCTGAGAGCTTATTTGCATGGTGCACAAGATGCTTACAGGGATTGGGAATTGTCCTACCCAAACACTACAAGTTTCGGGTTGTTCCTTGTCAAACCTGATAACCCATGGGAGtaG4-1BBL #2 (Mouse) (SEQ ID NO: 108)ATGGAAACTGATACACTCCTCCTGTGGGTCCTTCTTTTGTGGGTGCCCGGATCAACCGGCGATGGCTGGATGGCAGGCATCCCAGGACACCCAGGACACAACGGTACTCCAGGTCGAGACGGTCGGGATGGGACTCCTGGGGAGAAAGGCGAGAAAGGGGACGCTGGTTTGCTCGGTCCTAAGGGGGAAACCGGGGATGTAGGAATGACAGGGGCTGAAGGGCCTCGGGGATTTCCTGGGACACCAGGCAGGAAGGGTGAACCAGGGGAGGCCCTCCAGCGCACCGAGCCACGGCCAGCTCTGACCATAACAACAAGTCCAAACCTGGGCACACGCGAAAACAATGCTGACCAGGTGACTCCTGTAAGTCACATCGGATGCCCTAACACTACACAACAGGGCTCTCCTGTATTTGCAAAGCTTCTCGCAAAAAATCAAGCATCACTTTGTAATACAACCCTGAACTGGCATTCTCAGGACGGAGCAGGGTCCTCTTATTTGTCTCAAGGGCTCCGCTACGAAGAAGATAAAAAGGAATTGGTTGTTGACAGTCCAGGTTTGTATTATGTGTTTTTGGAACTTAAGCTGTCACCAACCTTCACTAACACCGGCCACAAGGTCCAAGGCTGGGTTAGTCTTGTTTTGCAAGCCAAACCTCAAGTGGATGATTTTGACAATCTGGCTTTGACTGTTGAGCTTTTTCCATGCAGTATGGAGAATAAACTGGTTGATCGGTCATGGTCACAGCTCCTTCTGCTCAAGGCCGGACATAGGCTGAGTGTGGGACTTCGGGCCTACTTGCACGGCGCCCAGGACGCATACCGAGACTGGGAACTCAGCTACCCTAACACAACTTCTTTTGGGTTGTTCCTTGTCAAAC CCGATAATCCTTGGGAAtaGHPGE2 #1 (Mouse) (SEQ ID NO: 109)ATGGAGACTGATACTTTGCTCCTGTGGGTTCTTCTCCTGTGGGTTCCTGGTTCCACAGGGGATATGCATGTCAATGGCAAGGTAGCACTCGTGACTGGGGCTGCACAGGGTATCGGGAAAGCTTTTGCCGAGGCCCTGTTGCTGCATGGCGCCAAGGTCGCTTTGGTAGATTGGAACTTGGAGGCTGGAGTTAAATGCAAAGCTGCACTCGACGAACAATTTGAGCCTCAAAAAACCCTCTTTGTGCAGTGTGACGTTGCTGACCAAAAGCAACTCAGGGACACATTCAGGAAGGTCGTAGACCATTTCGGACGCCTCGATATACTCGTTAATAATGCCGGGGTAAACAACGAAAAGAACTGGGAACAAACATTGCAAATCAACCTGGTAAGTGTCATTAGCGGAACTTATCTGGGTCTTGATTATATGAGCAAGCAGAACGGGGGCGAGGGCGGGATCATTATCAACATGTCAAGTCTTGCCGGATTGATGCCAGTTGCTCAGCAGCCTGTTTACTGTGCCAGCAAGCACGGTATTATTGGGTTTACCCGGAGTGCCGCCATGGCCGCAAATCTTATGAAGAGTGGGGTAAGACTGAATGTTATCTGCCCAGGTTTCGTAGATACCCCAATCCTGGAGAGCATCGAGAAGGAGGAAAATATGGGACAATACATTGAATATAAAGATCAAATCAAGGCTATGATGAAGTTCTACGGGGTTCTGCATCCATCCACAATTGCCAACGGGCTCATTAATCTGATTGAGGACGACGCCTTGAACGGAGCTATAATGAAAATCACAGCTTCCAAAGGCATTCACTTCCAAGATTATGATATATCACCCTTGCTTGTCAAGGCTCCTCTGACAAGT HPGE2 #2 (Mouse) (SEQ ID NO: 110)ATGCATGTCAATGGCAAGGTAGCACTCGTGACTGGGGCTGCACAGGGTATCGGGAAAGCTTTTGCCGAGGCCCTGTTGCTGCATGGCGCCAAGGTCGCTTTGGTAGATTGGAACTTGGAGGCTGGAGTTAAATGCAAAGCTGCACTCGACGAACAATTTGAGCCTCAAAAAACCCTCTTTGTGCAGTGTGACGTTGCTGACCAAAAGCAACTCAGGGACACATTCAGGAAGGTCGTAGACCATTTCGGACGCCTCGATATACTCGTTAATAATGCCGGGGTAAACAACGAAAAGAACTGGGAACAAACATTGCAAATCAACCTGGTAAGTGTCATTAGCGGAACTTATCTGGGTCTTGATTATATGAGCAAGCAGAACGGGGGCGAGGGCGGGATCATTATCAACATGTCAAGTCTTGCCGGATTGATGCCAGTTGCTCAGCAGCCTGTTTACTGTGCCAGCAAGCACGGTATTATTGGGTTTACCCGGAGTGCCGCCATGGCCGCAAATCTTATGAAGAGTGGGGTAAGACTGAATGTTATCTGCCCAGGTTTCGTAGATACCCCAATCCTGGAGAGCATCGAGAAGGAGGAAAATATGGGACAATACATTGAATATAAAGATCAAATCAAGGCTATGATGAAGTTCTACGGGGTTCTGCATCCATCCACAATTGCCAACGGGCTCATTAATCTGATTGAGGACGACGCCTTGAACGGAGCTATAATGAAAATCACAGCTTCCAAAGGCATTCACTTCCAAGATTATGATATATCACCCTTGCTTGTCAAG GCTCCTCTGACAAGT

Additional Embodiments

-   Provided below are enumerated paragraphs describing specific    embodiments:-   1. An engineered cell comprising:    -   a) a promoter; and    -   b) an exogenous polynucleotide sequence comprising an expression        cassette described in a formula, oriented from 5′ to 3′,        comprising        S1-E1-L-S2-E2        -   wherein        -   S1 comprises a polynucleotide sequence encoding a first            signal peptide,        -   E1 comprises a polynucleotide sequence encoding a first            effector molecule,        -   L comprises a linker polynucleotide sequence,        -   S2 comprises a polynucleotide sequence encoding a second            signal peptide,        -   E2 comprises a polynucleotide sequence encoding a second            effector molecule, and        -   wherein the promoter is operably linked to the expression            cassette, the first signal peptide is operably linked to the            first effector molecule, and the second signal peptide is            operably linked to the second effector molecule, and        -   wherein the engineered cell is selected from the group            consisting of: a mesenchymal stem cell (MSC), stem cell,            immune cell, natural killer (NK) cell, NKT cell, innate            lymphoid cell, tumor-infiltrating lymphocyte (TIL), mast            cell, eosinophil, basophil, monocyte, macrophage,            neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T            cell, CD4+ T cell, cytotoxic T lymphocyte (CTL),            viral-specific T cell, gamma-delta T cell, T regulatory            cell, and B cell.-   2. The engineered cell of paragraph 1, wherein the promoter    comprises an exogenous promoter polynucleotide sequence.-   3. The engineered cell of paragraph 1, wherein the promoter    comprises an endogenous promoter.-   4. The engineered cell of any one of paragraphs 1-3, wherein the    promoter is operably linked to the expression cassette such that the    polynucleotides are capable of being transcribed as a single    polynucleotide comprising the formula S1-E1-L-S2-E2.-   5. The engineered cell of paragraph 4, wherein the linker    polynucleotide sequence is operably associated with the translation    of the first effector molecule and the second effector molecule as    separate polypeptides.-   6. The engineered cell of paragraph 5, wherein the linker    polynucleotide sequence encodes a 2A ribosome skipping tag.-   7. The engineered cell of paragraph 6, wherein the 2A ribosome    skipping tag is selected from the group consisting of: P2A, T2A,    E2A, and F2A.-   8. The engineered cell of paragraph 5, wherein the linker    polynucleotide sequence encodes a T2A ribosome skipping tag.-   9. The engineered cell of paragraph 5, the linker polynucleotide    sequence encodes an Internal Ribosome Entry Site (IRES).-   10. The engineered cell of any one of paragraphs 5-9, wherein the    linker polynucleotide sequence encodes a cleavable polypeptide.-   11. The engineered cell of paragraph 10, wherein the cleavable    polypeptide comprises a Furin recognition polypeptide sequence.-   12. The engineered cell of any one of paragraphs 5-9, wherein the    linker polynucleotide sequence further encodes a Gly-Ser-Gly    polypeptide sequence.-   13. The engineered cell of any one of paragraphs 1-5, wherein the    linker polynucleotide sequence encodes a Furin recognition    polypeptide sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A    ribosome skipping tag in a Furin:Gly-Ser-Gly:T2A orientation from    N-terminus to C-terminus.-   14. The engineered cell of any one of paragraphs 1-3, wherein the    linker polynucleotide sequence encodes a second promoter,    -   wherein the promoter is operably linked to the expression        cassette such that a first polynucleotide comprising the formula        S1-E1 is capable of being transcribed,    -   wherein the second promoter is operably linked to the expression        cassette such that a second polynucleotide comprising the        formula S2-E2 is capable of being transcribed, and wherein the        first and the second polynucleotide are separate        polynucleotides.-   15. The engineered cell of paragraph 14, wherein the promoter and    the second promoter are identical.-   16. The engineered cell of paragraph 14, wherein the promoter and    the second promoter are different.-   17. The engineered cell of any one of paragraphs 1-16, wherein the    engineered cell is HLA-typed with reference to a subject in need of    therapeutic treatment.-   18. The engineered cell of any one of paragraphs 1-17, wherein the    engineered cell is a human cell.-   19. The engineered cell of paragraph 18, wherein the human cell is    an isolated cell from a subject.-   20. The engineered cell of paragraph 19, wherein the isolated cell    is isolated from a tissue consisting of the group of: bone marrow,    adipose tissue, the umbilical cord, fetal liver, muscle, and lung    tissue.-   21. The engineered cell of any one of paragraphs 1-20, wherein the    engineered cell is a cultured cell.-   22. The engineered cell of any one of paragraphs 1-21, wherein the    engineered MSC comprises a cellular marker phenotype comprising the    cellular markers CD105+, CD73+, and CD90+.-   23. The engineered cell of paragraph 22, wherein the cellular marker    phenotype further comprises a phenotype lacking or substantially    lacking one or more cellular markers selected from the group    consisting of: CD45, CD34, CD14, CD11b, CD79α, CD19, HLA class II,    and combinations thereof.-   24. The engineered cell of any one of paragraphs 1-21, wherein the    engineered MSC comprises a cellular marker phenotype comprising    CD105+, CD73+, CD90+, CD45−, CD34−, CD14−; a cellular marker    phenotype comprising CD105+, CD73+, CD90+, CD11b−, CD79α−; a    cellular marker phenotype comprising CD105+, CD73+, CD90+, CD19−,    HLA class II−; or a cellular marker phenotype comprising CD73+,    CD90+, CD105+, and CD166+, CD11b−, CD14−, CD19−, CD34−, CD45−, and    HLA−DR−.-   25. The engineered cell of any one of paragraphs 22-24, wherein the    cellular marker phenotype is determined or has been determined by    flow-cytometry.-   26. The engineered cell of any one of paragraphs 1-21, wherein the    engineered cell comprises a T cell.-   27. The engineered cell of any one of paragraphs 1-21, wherein the    engineered cell comprises a NK cell.-   28. The engineered cell of any one of paragraphs 1-21, wherein the    engineered cell comprises a NKT cell.-   29. The engineered cell of any of paragraphs 22-28, wherein the    cellular marker phenotype further comprises a cellular marker    comprising a cognate receptor or a cognate receptor ligand for the    first effector molecule, the second effector molecule, or the first    and second effector molecules expressed in the engineered cells.-   30. The engineered cell of paragraph 29, wherein the receptor is    selected from the group consisting of: IL12RB1, IL12RB2, CCL7, and    combinations thereof-   31. The engineered cell of any one of paragraphs 1-30, wherein the    promoter and/or the second promoter comprises a constitutive    promoter.-   32. The engineered cell of paragraph 31, wherein the constitutive    promoter is selected from the group consisting of: CMV, EFS, SFFV,    SV40, MND, PGK, UbC, hEFlaV1, hCAGG, hEF1aV2, hACTb, heIF4A1,    hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.-   33. The engineered cell of any one of paragraphs 1-30, wherein the    promoter comprises an SFFV promoter.-   34. The engineered cell of any one of paragraphs 1-30, wherein the    promoter and/or the second promoter comprises an inducible promoter.-   35. The engineered cell of paragraph 34, wherein the inducible    promoter is selected from the group consisting of: minP, NFkB    response element, CREB response element, NFAT response element, SRF    response element 1, SRF response element 2, AP1 response element,    TCF-LEF response element promoter fusion, Hypoxia responsive    element, SMAD binding element, STAT3 binding site, inducer molecule    responsive promoters, and tandem repeats thereof-   36. The engineered cell of any one of paragraphs 1-35, wherein the    first signal peptide or the second signal peptide comprises a native    signal peptide native to the first effector molecule or the second    effector molecule, respectively.-   37. The engineered cell of any one of paragraphs 1-36, wherein the    first signal peptide or the second signal peptide comprises a    non-native signal peptide non-native to the first effector molecule    or the second effector molecule, respectively.-   38. The engineered cell of paragraph 37, wherein the non-native    signal peptide is selected from the group consisting of: IL12, IL2,    optimized IL2, trypsiongen-2, Gaussia luciferase, CD5, human IgKVII,    murine IgKVII, VSV-G, prolactin, serum albumin preprotein,    azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2,    VEGFB, osteoprotegerin, serpin E1, GROalpha, CXCL12, and IL21.-   39. The engineered cell of any one of paragraphs 1-38, wherein the    first signal peptide and the second signal peptide are identical.-   40. The engineered cell of any one of paragraphs 1-39, wherein the    polynucleotide sequence encoding the first signal peptide comprises    a codon optimized polynucleotide sequence.-   41. The engineered cell of any one of paragraphs 1-40, wherein the    first secretion polypeptide is a human IL12 signal peptide.-   42. The engineered cell of any one of paragraphs 1-40, wherein the    polynucleotide sequence encoding the second signal peptide comprises    a codon optimized polynucleotide sequence.-   43. The engineered cell of any one of paragraphs 1-42, wherein the    second secretion polypeptide is a human IL21 signal peptide.-   44. The engineered cell of any one of paragraphs 1-42, wherein the    first effector molecule is selected from a therapeutic class,    wherein the therapeutic class is selected from the group consisting    of: a cytokine, a chemokine, a growth factor, a co-activation    molecule, a tumor microenvironment modifier a, a receptor, a ligand,    an antibody, a polynucleotide, a peptide, and an enzyme.-   45. The engineered cell of any one of paragraphs 1-44, wherein the    second effector molecule is selected from a therapeutic class,    wherein the therapeutic class is selected from the group consisting    of: a cytokine, a chemokine, a growth factor, a co-activation    molecule, a tumor microenvironment modifier, a receptor, a ligand,    an antibody, a polynucleotide, a peptide, and an enzyme.-   46. The engineered cell of paragraph 45, wherein the therapeutic    class of the first effector molecule and the second effector    molecule are different.-   47. The engineered cell of any one of paragraphs 1-46, wherein the    first effector molecule and/or the second effector molecule is a    modified effector molecule.-   48. The engineered cell of paragraph 47, wherein the first effector    molecule and/or the second effector molecule is modified to    comprises a cell membrane tethering domain.-   49. The engineered cell of paragraph 48, wherein the cell membrane    tethering domain comprises a transmembrane-intracellular domain or a    transmembrane domain.-   50. The engineered cell of paragraph 48, wherein the cell membrane    tethering domain comprises a cell surface receptor, or a cell    membrane-bound portion thereof-   51. The engineered cell of paragraph 50, wherein the modified    effector molecule is a fusion protein that comprises the cell    surface receptor, or a cell membrane-bound portion thereof-   52. The engineered cell of any one of paragraphs 48-51, wherein the    modified effector molecule further comprises a linker between the    effector molecule and the cell membrane tethering domain.-   53. The engineered cell of any one of paragraphs 47-52, wherein when    expressed the modified effector molecule is tethered to a cell    membrane of the engineered cell.-   54. The engineered cell of any one of paragraphs 44-53, wherein the    cytokine is selected from the group consisting of: IL12, IL7, IL21,    IL18, IL15, Type I interferons, and Interferon-gamma.-   55. The engineered cell of paragraph 54, wherein the IL12 cytokine    is an IL12p70 fusion protein.-   56. The engineered cell of any one of paragraphs 44-55, wherein the    chemokine is selected from the group consisting of: CCL21a, CXCL10,    CXCL11, CXCL13, CXCL10-11 fusion, CCL19, CXCL9, and XCL1.-   57. The engineered cell of any one of paragraphs 44-56, wherein the    growth factor is selected from the group consisting of: Flt3L and    GM-CSF.-   58. The engineered cell of any one of paragraphs 44-57, wherein the    co-activation molecule is selected from the group consisting of:    4-1BBL and CD40L.-   59. The engineered cell of any one of paragraphs 34-41, wherein the    tumor microenvironment modifier is selected from the group    consisting of: adenosine deaminase, TGFbeta inhibitors, immune    checkpoint inhibitors, VEGF inhibitors, and HPGE2.-   60. The engineered cell of paragraph 59, wherein the TGFbeta    inhibitors are selected from the group consisting of: an    anti-TGFbeta peptide, an anti-TGFbeta antibody, a TGFb-TRAP, and    combinations thereof-   61. The engineered cell of paragraph 59, wherein the immune    checkpoint inhibitors comprise anti-PD-1 antibodies.-   62. The engineered cell of paragraph 59, wherein the VEGF inhibitors    comprise anti-VEGF antibodies, anti-VEGF peptides, or combinations    thereof-   63. The engineered cell of any one of paragraphs 1-59, wherein the    first effector molecule and the second effector molecule are    human-derived effector molecules.-   64. The engineered cell of any one of paragraphs 1-63, wherein the    first effector molecule comprises IL12.-   65. The engineered cell of any one of paragraphs 1-63, wherein the    first effector molecule comprises an IL12p70 fusion protein.-   66. The engineered cell of paragraph 65, wherein the IL12p70 fusion    protein is a human IL12p70 fusion protein.-   67. The engineered cell of any one of paragraphs 64-66, wherein the    second effector molecule comprises CCL21a.-   68. The engineered cell of paragraph 67, wherein the CCL21a is a    human CCL21a.-   69. The engineered cell of any one of paragraphs 64-66, wherein the    second effector molecule comprises IL7.-   70. The engineered cell of paragraph 69, wherein the IL7 is a human    IL7.-   71. The engineered cell of any one of paragraphs 64-66, wherein the    second effector molecule comprises IL21.-   72. The engineered cell of paragraph 71, wherein the IL21 is a human    IL21.-   73. The engineered cell of any one of paragraphs 1-72, wherein the    expression cassette further comprises an E3 comprising a    polynucleotide sequence encoding a third effector molecule.-   74. The engineered cell of paragraph 73, wherein the third effector    molecule comprises Flt3L.-   75. The engineered cell of paragraph 73, wherein the third effector    molecule comprises anti-PD1.-   76. The engineered cell of paragraph 75, wherein the expression    cassette further comprises an E4 comprising a polynucleotide    sequence encoding a fourth effector molecule.-   77. The engineered cell of paragraph 76, wherein the fourth effector    molecule comprises adenosine deaminase.-   78. The engineered cell of paragraph 73, wherein the third effector    molecule comprises adenosine deaminase.-   79. The engineered cell of paragraph 73, wherein the third effector    molecule comprises CD40L.-   80. The engineered cell of paragraph 73, wherein the third effector    molecule comprises a CXCL10-CXCL11 fusion protein.-   81. The engineered cell of paragraph 73, wherein the third effector    molecule comprises XCL1.-   82. The engineered cell of paragraph 64, wherein the second effector    molecule comprises Flt3L.-   83. The engineered cell of paragraph 64, wherein the second effector    molecule comprises a CXCL10-CXCL11 fusion protein.-   84. The engineered cell of paragraph 64, wherein the second effector    molecule comprises anti-PD1.-   85. The engineered cell of paragraph 64, wherein the second effector    molecule comprises CD40L.-   86. The engineered cell of any one of paragraphs 1-63, wherein the    first effector molecule comprises interferon-beta and the second    effector molecule comprises Flt3L.-   87. The engineered cell of any one of paragraphs 1-86, wherein the    polynucleotide sequence encoding the first effector molecule    comprises a codon optimized polynucleotide sequence.-   88. The engineered cell of any one of paragraphs 1-87, wherein the    polynucleotide sequence encoding the second effector molecule    comprises a codon optimized polynucleotide sequence.-   89. The engineered cell of any one of paragraphs 1-88, wherein the    engineered cell comprises a polynucleotide sequence encoding the    promoter and the expression cassette.-   90. The engineered cell of paragraph 89, wherein the exogenous    polynucleotide sequence comprises the sequence shown in SEQ ID NO:    144.-   91. The engineered cell of any one of paragraphs 1-90, wherein the    exogenous polynucleotide sequence is integrated into the genome of    the engineered cell.-   92. The engineered cell of any one of paragraphs 1-91, wherein the    exogenous polynucleotide sequence comprises one or more viral vector    polynucleotide sequences.-   93. The engineered cell of paragraph 92, wherein the one or more    viral vector polynucleotide sequences comprise lentiviral,    retroviral, retrotransposon, or adenoviral polynucleotide sequences.-   94. The engineered cell of any one of paragraphs 1-93, wherein the    expression cassette further comprises following E2, an additional    exogenous polynucleotide sequence comprising a formula, oriented    from 5′ to 3′, comprising:    (L-S-E)_(X)    -   wherein    -   S comprises a polynucleotide sequence encoding a signal peptide,    -   E comprises a polynucleotide sequence encoding an effector        molecule,    -   L comprises a linker polynucleotide sequence,    -   X=1 to 20    -   wherein the promoter is operably linked to the expression        cassette, and wherein for each X the corresponding signal        peptide is operably associated with the effector molecule.-   95. An engineered cell comprising a construct, wherein the construct    comprises:    -   a) an SFFV promoter; and    -   b) an exogenous polynucleotide sequence comprising an expression        cassette described in a formula, oriented from 5′ to 3′,        comprising        S1-E1-L-S2-E2        -   wherein        -   S1 comprises a polynucleotide sequence encoding a first            signal peptide, wherein the first signal peptide is a human            IL12 signal peptide;        -   E1 comprises a polynucleotide sequence encoding a first            effector molecule, wherein the first effector molecule is a            human IL12p70 fusion protein;        -   L comprises a linker polynucleotide sequence, wherein the            linker polynucleotide sequence encodes a Furin recognition            polypeptide sequence, a Gly-Ser-Gly polypeptide sequence,            and a T2A ribosome skipping tag in a Furin:Gly-Ser-Gly:T2A            orientation from N-terminus to C-terminus;        -   S2 comprises a polynucleotide sequence encoding a second            signal peptide, wherein the second signal peptide is a human            IL21 signal peptide;        -   E2 comprises a polynucleotide sequence encoding a second            effector molecule, wherein the second effector molecule is            human IL21; and        -   wherein the SFFV promoter is operably linked to the            expression cassette, the first signal peptide is operably            linked to the first effector molecule, and the second signal            peptide is operably linked to the second effector molecule,            and        -   wherein the engineered cell is selected from the group            consisting of: a mesenchymal stem cell (MSC), stem cell,            immune cell, natural killer (NK) cell, NKT cell, innate            lymphoid cell, tumor-infiltrating lymphocyte (TIL), mast            cell, eosinophil, basophil, monocyte, macrophage,            neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T            cell, CD4+ T cell, cytotoxic T lymphocyte (CTL),            viral-specific T cell, gamma-delta T cell, T regulatory            cell, and B cell.-   96. The engineered cell of paragraph 95, wherein the construct    comprises the polynucleotide sequence shown in SEQ ID NO: 144.-   97. The engineered cell of paragraph 95 or paragraph 96, wherein the    engineered cell is HLA-typed with reference to a subject in need of    therapeutic treatment.-   98. The engineered cell of any one of paragraphs 95-97, wherein the    engineered cell is a human cell.-   99. The engineered cell of paragraph 98, wherein the human cell is    an isolated cell from a subject.-   100. The engineered cell of paragraph 99, wherein the isolated cell    is isolated from a tissue consisting of the group of: bone marrow,    adipose tissue, the umbilical cord, fetal liver, muscle, and lung    tissue.-   101. The engineered cell of any one of paragraphs 95-100, wherein    the engineered cell is a cultured cell.-   102. The engineered cell of any one of paragraphs 95-101, wherein    the engineered MSC comprises a cellular marker phenotype comprising    the cellular markers CD105+, CD73+, and CD90+.-   103. The engineered cell of paragraph 102, wherein the cellular    marker phenotype further comprises a phenotype lacking or    substantially lacking one or more cellular markers selected from the    group consisting of: CD45, CD34, CD14, CD11b, CD79α, CD19, HLA class    II, and combinations thereof-   104. The engineered cell of any one of paragraphs 95-101, wherein    the engineered MSC comprises a cellular marker phenotype comprising    CD105+, CD73+, CD90+, CD45−, CD34−, CD14−; a cellular marker    phenotype comprising CD105+, CD73+, CD90+, CD11b−, CD79α−; a    cellular marker phenotype comprising CD105+, CD73+, CD90+, CD19−,    HLA class II−; or a cellular marker phenotype comprising CD73+,    CD90+, CD105+, and CD166+, CD11b−, CD14−, CD19−, CD34−, CD45−, and    HLA−DR−.-   105. The engineered cell of any one of paragraphs 95-101, wherein    the engineered cell comprises a T cell.-   106. The engineered cell of paragraph 105, wherein the T cell is a    CD8+ T cell, a CD4+ T cell, a cytotoxic T lymphocyte (CTL), a    viral-specific T cell, a gamma-delta T cell, or a T regulatory cell.-   107. The engineered cell of any one of paragraphs 95-101, wherein    the engineered cell comprises a NK cell.-   108. The engineered cell of any one of paragraphs 95-101, wherein    the engineered cell comprises a NKT cell.-   109. The engineered cell of any one of paragraphs 95-101, wherein    the engineered cell comprises a monocyte cell.-   110. The engineered cell of any one of paragraphs 95-101, wherein    the engineered cell comprises a macrophage.-   111. The engineered cell of any one of paragraphs 95-101, wherein    the engineered cell comprises a TIL.-   112. The engineered cell of any one of paragraphs 95-111, wherein    the exogenous polynucleotide sequence is integrated into the genome    of the engineered cell.-   113. The engineered cell of any one of paragraphs 95-112, wherein    the exogenous polynucleotide sequence comprises one or more viral    vector polynucleotide sequences.-   114. The engineered cell of paragraph 113, wherein the one or more    viral vector polynucleotide sequences comprise lentiviral,    retroviral, retrotransposon, or adenoviral polynucleotide sequences.-   115. The engineered cell of paragraph 113, wherein the one or more    viral vector polynucleotide sequences comprise lentiviral    polynucleotide sequences.-   116. The engineered cell of any one of paragraphs 1-115, wherein the    cell secretes each effector molecule.-   117. The engineered cell of paragraph 116, wherein the first    effector molecule is secreted at a ratio that is 10 fold higher    relative to secretion of the second effector molecule.-   118. The engineered cell of any one of paragraphs 1-117, wherein the    cell further comprises an antigen recognizing receptor.-   119. The engineered cell of paragraph 118, wherein the antigen    recognizing receptor recognizes an antigen selected from the group    consisting of: 5T4, ADAM9, ADGRE2, AFP, AXL, B7-H3, B7-H4, B7-H6,    C4.4, CA6, Cadherin 3, Cadherin 6, CCR1, CCR4, CD117, CD123, CD131,    CD133, CD138, CD142, CD166, CD25, CD244, CD30, CD300LF, CD33, CD352,    CD37, CD38, CD44, CD56, CD66e, CD70, CD71, CD74, CD79b, CD80, CD93,    CEA, CEACAM5, Claudin18.2, CLEC12A, cMet, CSPG4, CTLA, DLK1, DLL3,    DR5, EGFR, EMB, ENPP3, EpCAM, EphA2, Ephrin A4, ETBR, FGFR2, FGFR3,    FRalpha, FRb, FLT3, GAPT, GCC, GD2, GFRa4, gpA33, GPC3, gpNBM,    GPRC5, HER2, IL-1RAP, IL-13R, IL-13Ra, IL-13Ra2, IL-8, IL-15,    IL1RAP, Integrin aV, KIT, L1CAM, LAMP1, LAT2, Lewis Y, LeY, LILRA2,    LILRB2, LIV-1, LRRC, LY6E, MCSP, Mesothelin, MLC1, MS4A3, MUC1,    MUC16, MUC1C, MYADM, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY ESO 1,    Ovarin, P-cadherin, pan-Erb2, PIEZO1, PRAM1, PSCA, PSMA, PTK7, ROR1,    S Aures, SCT, SLAMF7, SLC22A16, SLC17A9, SLITRK6, SPNS3, SSTR2,    STEAP1, Survivin, TDGF1, TIM1, TROP2, VSTM1, and WT1-   120. The engineered cell of paragraph 118 or paragraph 119, wherein    the antigen recognizing receptor comprises an antigen-binding    domain.-   121. The engineered cell of paragraph 120, wherein the    antigen-binding domain comprises an antibody, an antigen-binding    fragment of an antibody, a F(ab) fragment, a F(ab′) fragment, a    single chain variable fragment (scFv), or a single-domain antibody    (sdAb).-   122. The engineered cell of paragraph 120, wherein the    antigen-binding domain comprises a single chain variable fragment    (scFv).-   123. The engineered cell of paragraph 122, wherein the scFv    comprises a heavy chain variable domain (VH) and a light chain    variable domain (VL).-   124. The engineered cell of paragraph 123, wherein the VH and VL are    separated by a peptide linker.-   125. The engineered cell of paragraph 124, wherein the scFv    comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy    chain variable domain, L is the peptide linker, and VL is the light    chain variable domain.-   126. The engineered cell of any one of paragraphs 118-125, the    antigen recognizing receptor is a chimeric antigen receptor (CAR) or    T cell receptor (TCR).-   127. The engineered cell of any one of paragraphs 118-125, the    antigen recognizing receptor is a chimeric antigen receptor (CAR).-   128. The engineered cell of paragraph 127, wherein the CAR comprises    one or more intracellular signaling domains, and the one or more    intracellular signaling domains are selected from the group    consisting of: a CD3zeta-chain intracellular signaling domain, a    CD97 intracellular signaling domain, a CD11a-CD18 intracellular    signaling domain, a CD2 intracellular signaling domain, an ICOS    intracellular signaling domain, a CD27 intracellular signaling    domain, a CD154 intracellular signaling domain, a CD8 intracellular    signaling domain, an OX40 intracellular signaling domain, a 4-1BB    intracellular signaling domain, a CD28 intracellular signaling    domain, a ZAP40 intracellular signaling domain, a CD30 intracellular    signaling domain, a GITR intracellular signaling domain, an HVEM    intracellular signaling domain, a DAP10 intracellular signaling    domain, a DAP12 intracellular signaling domain, and a MyD88    intracellular signaling domain.-   129. The engineered cell of paragraph 127 or paragraph 128, wherein    the CAR comprises a transmembrane domain, and the transmembrane    domain is selected from the group consisting of: a CD8 transmembrane    domain, a CD28 transmembrane domain a CD3zeta-chain transmembrane    domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an    OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4    transmembrane domain, a PD-1 transmembrane domain, a LAG-3    transmembrane domain, a 2B4 transmembrane domain, and a BTLA    transmembrane domain.-   130. The engineered cell of any one of paragraphs 127-129, wherein    the CAR comprises a spacer region between the antigen-binding domain    and the transmembrane domain.-   131. A population of cells, the population of cells comprising any    of the engineered cells of any one of paragraphs 1-130.-   132. The population of cells of paragraph 131, wherein the    population of cells is enriched for the engineered cells.-   133. The population of cells of paragraph 131 or paragraph 132,    wherein the first effector molecule, the second effector molecule,    or the first and second effector molecules expressed in the    engineered cells promotes increased growth, viability, or growth and    viability relative to cells in the population that do not express    the first effector molecule, the second effector molecule, or the    first and second effector molecules.-   134. The population of cells of paragraph 133, wherein the first    effector molecule is IL12 or an IL12p70 fusion protein.-   135. The population of cells of paragraph 134, wherein the    population of cells enriched for the engineered cells express IL12    receptor β1 or increased levels thereof, IL12 receptor β2 or    increased levels thereof, or IL12 receptor β1 and IL12 receptor β2    or increased levels thereof-   136. The population of cells of any of paragraphs 133-135, wherein    the second effector molecule is IL21.-   137. The population of cells of any of paragraphs 133-135, wherein    the second effector molecule is CCL21.-   138. The population of cells of paragraph 137, wherein the    population of cells enriched for the engineered cells express a    CCL21 receptor or increased levels thereof-   139. The population of cells of paragraph 138, wherein the CCL21    receptor is CCR7.-   140. A method of stimulating a cell-mediated immune response to a    tumor cell in a subject, the method comprising administering to a    subject having a tumor a therapeutically effective dose of any of    the engineered cells of any one of paragraphs 1-114 or the    population of cells of any of paragraphs 131-139.-   141. A method of providing an anti-tumor immunity in a subject, the    method comprising administering to a subject in need thereof a    therapeutically effective dose of any of the engineered cells of any    one of paragraphs 1-114 or the population of cells of any of    paragraphs 131-139.-   142. A method of treating a subject having cancer, the method    comprising administering to a subject having a tumor a    therapeutically effective dose of any of the engineered cell of any    one of paragraphs 1-114 or the population of cells of any of    paragraphs 131-139.-   143. A method of reducing tumor volume in a subject, the method    comprising administering to a subject having a tumor a    therapeutically effective dose of any of the engineered cells of any    one of paragraphs 1-114 or the population of cells of any of    paragraphs 131-139.-   144. The method of any one of paragraphs 140-143, wherein the    engineered cell is derived from the subject.-   145. The method of any one of paragraphs 140-143, wherein the    engineered cell is allogeneic with reference to the subject.-   146. The method of any one of paragraphs 140-145, wherein the tumor    is selected from the group consisting of: an adenocarcinoma, an    acute myeloid leukemia (AML), an acute lymphoblastic B-cell leukemia    (BALL), an acute lymphoblastic T-cell leukemia (TALL), a B-cell    prolymphocytic leukemia, a bladder tumor, a brain tumor, a breast    tumor, a cervical tumor, a chronic lymphocytic leukemia, a chronic    myeloid leukemia (CML), a colorectal tumor, an esophageal tumor, a    glioma, a kidney tumor, a liver tumor, a lung tumor, a lymphoma, a    melanoma, a mesothelioma, a myelodysplasia, an ovarian tumor, a    pancreatic tumor, a plasma cell myeloma, a prostate tumor, a skin    tumor, a thyroid tumor, and a uterine tumor.-   147. The method of any one of paragraphs 140-145, wherein the tumor    is an ovarian tumor.-   148. The method of any one of paragraphs 140-147, wherein the tumor    is a tumor located in a peritoneal space.-   149. An engineered cell comprising:

a) a promoter; and

b) an exogenous polynucleotide sequence comprising an expressioncassette described in a formula, oriented from 5′ to 3′, comprising:(L-S-E)_(X)

-   -   -   wherein        -   S comprises a polynucleotide sequence encoding a signal            peptide,        -   E comprises a polynucleotide sequence encoding an effector            molecule,        -   L comprises a linker polynucleotide sequence,        -   X=2 to 20,        -   wherein the promoter is operably linked to the expression            cassette, wherein for the first iteration of the (L-S-E)            unit L is absent, and wherein for each X the corresponding            signal peptide is operably associated with the effector            molecule, and        -   wherein the engineered cell is selected from the group            consisting of: a mesenchymal stem cell (MSC), stem cell,            immune cell, natural killer (NK) cell, NKT cell, innate            lymphoid cell, tumor-infiltrating lymphocyte (TIL), mast            cell, eosinophil, basophil, monocyte, macrophage,            neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T            cell, CD4+ T cell, cytotoxic T lymphocyte (CTL),            viral-specific T cell, gamma-delta T cell, T regulatory            cell, and B cell.

-   150. A population of cells comprising one or more engineered cells,    wherein the one or more engineered cells comprise:

a) a promoter; and

b) an exogenous polynucleotide sequence comprising an expressioncassette described in a formula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2

-   -   -   wherein        -   S1 comprises a polynucleotide sequence encoding a first            signal peptide,        -   E1 comprises a polynucleotide sequence encoding a first            effector molecule,        -   L comprises a linker polynucleotide sequence,        -   S2 comprises a polynucleotide sequence encoding a second            signal peptide,        -   E2 comprises a polynucleotide sequence encoding a second            effector molecule, and        -   wherein the promoter is operably linked to the expression            cassette, the first signal peptide is operably linked to the            first effector molecule, and the second signal peptide is            operably linked to the second effector molecule, and        -   wherein the engineered cell is selected from the group            consisting of: a mesenchymal stem cell (MSC), stem cell,            immune cell, natural killer (NK) cell, NKT cell, innate            lymphoid cell, tumor-infiltrating lymphocyte (TIL), mast            cell, eosinophil, basophil, monocyte, macrophage,            neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T            cell, CD4+ T cell, cytotoxic T lymphocyte (CTL),            viral-specific T cell, gamma-delta T cell, T regulatory            cell, and B cell.

-   151. A population of cells comprising one or more engineered cells,    wherein the one or more engineered cells comprise:    -   a) a promoter; and    -   b) an exogenous polynucleotide sequence comprising an expression        cassette described in a formula, oriented from 5′ to 3′,        comprising        S1-E1-L-S2-E2        -   wherein        -   S1 comprises a polynucleotide sequence encoding a first            signal peptide,        -   E1 comprises a polynucleotide sequence encoding a first            effector molecule,        -   L comprises a linker polynucleotide sequence,        -   S2 comprises a polynucleotide sequence encoding a second            signal peptide,        -   E2 comprises a polynucleotide sequence encoding a second            effector molecule, and        -   wherein the promoter is operably linked to the expression            cassette, the first signal peptide is operably linked to the            first effector molecule, and the second signal peptide is            operably linked to the second effector molecule, and        -   wherein the first effector molecule, the second effector            molecule, or the first and second effector molecules            expressed in the engineered cells promotes increased growth,            viability, or growth and viability relative to cells in the            population that do not express the first effector molecule,            the second effector molecule, or the first and second            effector molecules, and        -   wherein the engineered cell is selected from the group            consisting of: a mesenchymal stem cell (MSC), stem cell,            immune cell, natural killer (NK) cell, NKT cell, innate            lymphoid cell, tumor-infiltrating lymphocyte (TIL), mast            cell, eosinophil, basophil, monocyte, macrophage,            neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T            cell, CD4+ T cell, cytotoxic T lymphocyte (CTL),            viral-specific T cell, gamma-delta T cell, T regulatory            cell, and B cell.

-   152. The population of cells of paragraph 151, wherein the one or    more engineered cells express a cognate receptor or cognate receptor    ligand for the first effector molecule, the second effector    molecule, or the first and second effector molecules expressed in    the engineered cells.

-   153. The population of cells of paragraph 151 or paragraph 152,    wherein the first effector molecule is IL12 or an IL12p70 fusion    protein.

-   154. The population of cells of any of paragraphs 151-153, wherein    the second effector molecule is IL21.

-   155. The population of cells of any of paragraphs 151-153, wherein    the second effector molecule is CCL21.

-   156. A population of cells comprising one or more engineered cells,    wherein the one or more engineered cells comprise a construct,    wherein the construct comprises:    -   a) an SFFV promoter; and    -   b) an exogenous polynucleotide sequence comprising an expression        cassette described in a formula, oriented from 5′ to 3′,        comprising        S1-E1-L-S2-E2        -   wherein        -   S1 comprises a polynucleotide sequence encoding a first            signal peptide, wherein the first signal peptide is a human            IL12 signal peptide;        -   E1 comprises a polynucleotide sequence encoding a first            effector molecule, wherein the first effector molecule is a            human IL12p70 fusion protein;        -   L comprises a linker polynucleotide sequence, wherein the            linker polynucleotide sequence encodes a Furin recognition            polypeptide sequence, a Gly-Ser-Gly polypeptide sequence,            and a T2A ribosome skipping tag in a Furin:Gly-Ser-Gly:T2A            orientation from N-terminus to C-terminus;        -   S2 comprises a polynucleotide sequence encoding a second            signal peptide, wherein the second signal peptide is a human            IL21 signal peptide;        -   E2 comprises a polynucleotide sequence encoding a second            effector molecule, wherein the second effector molecule is            human IL21; and        -   wherein the SFFV promoter is operably linked to the            expression cassette, the first signal peptide is operably            linked to the first effector molecule, and the second signal            peptide is operably linked to the second effector molecule,            and        -   wherein the engineered cell is selected from the group            consisting of: a mesenchymal stem cell (MSC), stem cell,            immune cell, natural killer (NK) cell, NKT cell, innate            lymphoid cell, tumor-infiltrating lymphocyte (TIL), mast            cell, eosinophil, basophil, monocyte, macrophage,            neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T            cell, CD4+ T cell, cytotoxic T lymphocyte (CTL),            viral-specific T cell, gamma-delta T cell, T regulatory            cell, and B cell.

-   157. A population of cells comprising one or more engineered cells,    wherein the one or more engineered cells comprise a construct,    wherein the construct comprises:    -   a) an SFFV promoter; and    -   b) an exogenous polynucleotide sequence comprising an expression        cassette described in a formula, oriented from 5′ to 3′,        comprising        S1-E1-L-S2-E2        -   wherein        -   S1 comprises a polynucleotide sequence encoding a first            signal peptide, wherein the first signal peptide is a human            IL12 signal peptide;        -   E1 comprises a polynucleotide sequence encoding a first            effector molecule, wherein the first effector molecule is a            human IL12p70 fusion protein;        -   L comprises a linker polynucleotide sequence, wherein the            linker polynucleotide sequence encodes a Furin recognition            polypeptide sequence, a Gly-Ser-Gly polypeptide sequence,            and a T2A ribosome skipping tag in a Furin:Gly-Ser-Gly:T2A            orientation from N-terminus to C-terminus;        -   S2 comprises a polynucleotide sequence encoding a second            signal peptide, wherein the second signal peptide is a human            IL21 signal peptide;        -   E2 comprises a polynucleotide sequence encoding a second            effector molecule, wherein the second effector molecule is            human IL21; and        -   wherein the SFFV promoter is operably linked to the            expression cassette, the first signal peptide is operably            linked to the first effector molecule, and the second signal            peptide is operably linked to the second effector molecule,            and        -   wherein the first effector molecule, the second effector            molecule, or the first and second effector molecules            expressed in the engineered cells promotes increased growth,            viability, or growth and viability relative to cells in the            population that do not express the first effector molecule,            the second effector molecule, or the first and second            effector molecules, and        -   wherein the engineered cell is selected from the group            consisting of: a mesenchymal stem cell (MSC), stem cell,            immune cell, natural killer (NK) cell, NKT cell, innate            lymphoid cell, tumor-infiltrating lymphocyte (TIL), mast            cell, eosinophil, basophil, monocyte, macrophage,            neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T            cell, CD4+ T cell, cytotoxic T lymphocyte (CTL),            viral-specific T cell, gamma-delta T cell, T regulatory            cell, and B cell.

-   158. The population of cells of paragraph 156 or paragraph 157,    wherein the construct comprises the polynucleotide sequence shown in    SEQ ID NO: 144.

-   159. A method of producing a population of cells enriched for one or    more receptors or receptor ligands, comprising culturing one or more    cells under conditions where the one or more cells are contacted    with a first effector molecule, a second effector molecule, or a    first and a second effector molecule, wherein the contacted cells    express one or more cognate receptors or cognate receptor ligands    for the first effector molecule, the second effector molecule, or    the first and second effector molecules, and wherein the first    effector molecule, the second effector molecule, or the first and    the second effector molecules increase growth, viability, or growth    and viability of the contacted cells relative to cells cultured in    the absence of the first effector molecule, the second effector    molecule, or the first and second effector molecules.

-   160. The method of paragraph 159, wherein the first effector    molecule, the second effector molecule, or the first and second    effector molecules are heterologously expressed in one or more    cells, and the one or more cells are contacted with the first    effector molecule, the second effector molecule, or the first and    second effector molecules in an autocrine manner.

-   161. The method of paragraph 159, wherein the first effector    molecule, the second effector molecule, or the first and second    effector molecules are expressed in one or more additional cells,    and the one or more cells are contacted with the first effector    molecule, the second effector molecule, or the first and second    effector molecules in an paracrine manner.

-   162. The method of paragraph 161, wherein the one or more additional    cells are a feeder cells.

-   163. The method of paragraph 159, wherein the one or more cells are    cultured in media.

-   164. The method of paragraph 163, wherein the one or more cells are    contacted with the first effector molecule, the second effector    molecule, or the first and second effector molecules by addition of    a soluble first effector molecule, a soluble second effector    molecule, or a soluble first and second effector molecules to the    media.

-   165. The method of paragraph 163 or paragraph 164, wherein the    soluble first effector molecule and/or soluble second effector    molecule is a recombinant effector molecule.

-   166. The method of paragraph 159, wherein the one or more cells are    cultured under adherent conditions.

-   167. The method of paragraph 166, wherein the one or more cells are    adhered onto a surface.

-   168. The method of paragraph 167, wherein the adhered cells are    contacted with the first effector molecule, the second effector    molecule, or the first and second effector molecules by exposing the    one or more cells to first effector molecule, the second effector    molecule, or the first and second effector molecules is immobilized    on the surface.

-   169. The method of any one of paragraphs 159-168, wherein the first    effector molecule is IL12 or an IL12p70 fusion protein.

-   170. The method of paragraph 169, wherein the population of cells is    enriched for IL12 receptor β1 (IL12Rβ1), enriched for IL12 receptor    β2 (IL12Rβ2), or enriched for IL12Rβ1 and IL12Rβ2.

-   171. The method of paragraph 170, wherein the population of MSCs    comprises a cellular marker phenotype comprising the cellular    markers CD105+, CD73+, CD90+, IL12Rβ1+, and IL12Rβ2+.

-   172. The method of paragraph 171, wherein the cellular marker    phenotype further comprises a phenotype lacking or substantially    lacking one or more cellular markers selected from the group    consisting of: CD45, CD34, CD14, CD11b, CD79α, CD19, HLA class II,    and combinations thereof

-   173. The method of paragraph 159, wherein the population of cells    comprises a cell selected from the group consisting of: natural    killer (NK) cells, NKT cells, innate lymphoid cells, mast cells,    eosinophils, basophils, monocytes, macrophages, neutrophils, and    dendritic cells, T cells, CD8+ T cells, CD4+ T cells, gamma-delta T    cells, and T regulatory cells, and B cells.

-   174. The method of paragraph 173, wherein the population of cells    comprises a T cell, a NK cell, a NKT cell, a monocyte, a macrophage,    or a myeloid derived cell.

-   175. The method of any one of paragraphs 159-174, wherein the second    effector molecule is IL21.

-   176. The method of any one of paragraphs 159-174, wherein the second    effector molecule is CCL21.

-   177. The method of paragraph 176, wherein the population of cells is    enriched for CCR7.

-   178. The method of paragraph 177, wherein the population of MSCs    comprises a cellular marker phenotype comprising the cellular    markers CD105+, CD73+, CD90+, IL12Rβ1+, IL12Rβ2+, and CCR7+.

-   179. The method of paragraph 178, wherein the cellular marker    phenotype further comprises a phenotype lacking or substantially    lacking one or more cellular markers selected from the group    consisting of: CD45, CD34, CD14, CD11b, CD79α, CD19, HLA class II,    and combinations thereof

-   180. A population of cells enriched for one or more receptors or    receptor ligands produced by the method of any one of paragraphs    159-179.

-   181. An exogenous polynucleotide sequence comprising a promoter and    an expression cassette described in a formula, oriented from 5′ to    3′, comprising    S1-E1-L-S2-E2    -   wherein    -   S1 comprises a polynucleotide sequence encoding a first signal        peptide,    -   E1 comprises a polynucleotide sequence encoding a first effector        molecule,    -   L comprises a linker polynucleotide sequence,    -   S2 comprises a polynucleotide sequence encoding a second signal        peptide,    -   E2 comprises a polynucleotide sequence encoding a second        effector molecule, and    -   wherein the promoter is operably linked to the expression        cassette, the first signal peptide is operably linked to the        first effector molecule, and the second signal peptide is        operably linked to the second effector molecule.

-   182. The exogenous polynucleotide sequence of paragraph 181, wherein    the promoter comprises an exogenous promoter polynucleotide    sequence.

-   183. The exogenous polynucleotide sequence of paragraph 181, wherein    the promoter comprises an endogenous promoter.

-   184. The exogenous polynucleotide sequence of any one of paragraphs    181-183, wherein the promoter is operably linked to the expression    cassette such that the polynucleotides are capable of being    transcribed as a single polynucleotide comprising the formula    S1-E1-L-S2-E2.

-   185. The exogenous polynucleotide sequence of paragraph 184, wherein    the linker polynucleotide sequence is operably associated with the    translation of the first effector molecule and the second effector    molecule as separate polypeptides.

-   186. The exogenous polynucleotide sequence of paragraph 185, wherein    the linker polynucleotide sequence encodes a 2A ribosome skipping    tag.

-   187. The exogenous polynucleotide sequence of paragraph 186, wherein    the 2A ribosome skipping tag is selected from the group consisting    of: P2A, T2A, E2A, and F2A.

-   188. The exogenous polynucleotide sequence of paragraph 185, wherein    the linker polynucleotide sequence encodes a T2A ribosome skipping    tag.

-   189. The exogenous polynucleotide sequence of paragraph 185, the    linker polynucleotide sequence encodes an Internal Ribosome Entry    Site (IRES).

-   190. The exogenous polynucleotide sequence of any one of paragraphs    185-189, wherein the linker polynucleotide sequence encodes a    cleavable polypeptide.

-   191. The exogenous polynucleotide sequence of paragraph 190, wherein    the cleavable polypeptide comprises a Furin recognition polypeptide    sequence.

-   192. The exogenous polynucleotide sequence of any one of paragraphs    185-191, wherein the linker polynucleotide sequence further encodes    a Gly-Ser-Gly polypeptide sequence.

-   193. The exogenous polynucleotide sequence of any one of paragraphs    181-185, wherein the linker polynucleotide sequence encodes a Furin    recognition polypeptide sequence, a Gly-Ser-Gly polypeptide    sequence, and a T2A ribosome skipping tag in a Furin:Gly-Ser-Gly:T2A    orientation from N-terminus to C-terminus.

-   194. The exogenous polynucleotide sequence of any one of paragraphs    181-183, wherein the linker polynucleotide sequence encodes a second    promoter,    -   wherein the promoter is operably linked to the expression        cassette such that a first polynucleotide comprising the formula        S1-E1 is capable of being transcribed,    -   wherein the second promoter is operably linked to the expression        cassette such that a second polynucleotide comprising the        formula S2-E2 is capable of being transcribed, and wherein the        first and the second polynucleotide are separate        polynucleotides.

-   195. The exogenous polynucleotide sequence of paragraph 194, wherein    the promoter and the second promoter are identical.

-   196. The exogenous polynucleotide sequence of paragraph 194, wherein    the promoter and the second promoter are different.

-   197. The exogenous polynucleotide sequence of any one of paragraphs    181-196, wherein the promoter and/or the second promoter comprises a    constitutive promoter.

-   198. The exogenous polynucleotide sequence of paragraph 197, wherein    the constitutive promoter is selected from the group consisting of:    CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb,    heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

-   199. The exogenous polynucleotide sequence of any one of paragraphs    181-196, wherein the promoter comprises an SFFV promoter.

-   200. The exogenous polynucleotide sequence of any one of paragraphs    181-196, wherein the promoter and/or the second promoter comprises    an inducible promoter.

-   201. The exogenous polynucleotide sequence of paragraph 200, wherein    the inducible promoter is selected from the group consisting of:    minP, NFkB response element, CREB response element, NFAT response    element, SRF response element 1, SRF response element 2, AP1    response element, TCF-LEF response element promoter fusion, Hypoxia    responsive element, SMAD binding element, STAT3 binding site,    inducer molecule responsive promoters, and tandem repeats thereof

-   202. The exogenous polynucleotide sequence of any one of paragraphs    181-201, wherein the first signal peptide or the second signal    peptide comprises a native signal peptide native to the first    effector molecule or the second effector molecule, respectively.

-   203. The exogenous polynucleotide sequence of any one of paragraphs    181-202, wherein the first signal peptide or the second signal    peptide comprises a non-native signal peptide non-native to the    first effector molecule or the second effector molecule,    respectively.

-   204. The exogenous polynucleotide sequence of paragraph 203, wherein    the non-native signal peptide is selected from the group consisting    of: IL12, IL2, optimized IL2, trypsiongen-2, Gaussia luciferase,    CD5, human IgKVII, murine IgKVII, VSV-G, prolactin, serum albumin    preprotein, azurocidin preprotein, osteonectin, CD33, IL6, IL8,    CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROalpha, CXCL12,    and IL21.

-   205. The exogenous polynucleotide sequence of any one of paragraphs    181-204, wherein the first signal peptide and the second signal    peptide are identical.

-   206. The exogenous polynucleotide sequence of any one of paragraphs    181-205, wherein the polynucleotide sequence encoding the first    signal peptide comprises a codon optimized polynucleotide sequence.

-   207. The exogenous polynucleotide sequence of any one of paragraphs    181-206, wherein the first secretion polypeptide is a human IL12    signal peptide.

-   208. The exogenous polynucleotide sequence of any one of paragraphs    181-206, wherein the polynucleotide sequence encoding the second    signal peptide comprises a codon optimized polynucleotide sequence.

-   209. The exogenous polynucleotide sequence of any one of paragraphs    181-208, wherein the second secretion polypeptide is a human IL21    signal peptide.

-   210. The exogenous polynucleotide sequence of any one of paragraphs    181-208, wherein the first effector molecule is selected from a    therapeutic class, wherein the therapeutic class is selected from    the group consisting of: a cytokine, a chemokine, a growth factor, a    co-activation molecule, a tumor microenvironment modifier a, a    receptor, a ligand, an antibody, a polynucleotide, a peptide, and an    enzyme.

-   211. The exogenous polynucleotide sequence of any one of paragraphs    181-210, wherein the second effector molecule is selected from a    therapeutic class, wherein the therapeutic class is selected from    the group consisting of: a cytokine, a chemokine, a growth factor, a    co-activation molecule, a tumor microenvironment modifier, a    receptor, a ligand, an antibody, a polynucleotide, a peptide, and an    enzyme.

-   212. The exogenous polynucleotide sequence of paragraph 211, wherein    the therapeutic class of the first effector molecule and the second    effector molecule are different.

-   213. The exogenous polynucleotide sequence of any one of paragraphs    181-212, wherein the first effector molecule and/or the second    effector molecule is a modified effector molecule.

-   214. The exogenous polynucleotide sequence of paragraph 213, wherein    the first effector molecule and/or the second effector molecule is    modified to comprises a cell membrane tethering domain.

-   215. The exogenous polynucleotide sequence of paragraph 214, wherein    the cell membrane tethering domain comprises a    transmembrane-intracellular domain or a transmembrane domain.

-   216. The exogenous polynucleotide sequence of paragraph 214, wherein    the cell membrane tethering domain comprises a cell surface    receptor, or a cell membrane-bound portion thereof

-   217. The exogenous polynucleotide sequence of paragraph 216, wherein    the modified effector molecule is a fusion protein that comprises    the cell surface receptor, or a cell membrane-bound portion thereof

-   218. The exogenous polynucleotide sequence of any one of paragraphs    214-217, wherein the modified effector molecule further comprises a    linker between the effector molecule and the cell membrane tethering    domain.

-   219. The exogenous polynucleotide sequence of any one of paragraphs    213-218, wherein when expressed in a cell, the modified effector    molecule is tethered to a cell membrane of the cell.

-   220. The exogenous polynucleotide sequence of any one of paragraphs    210-219 wherein the cytokine is selected from the group consisting    of: IL12, IL7, IL21, IL18, IL15, Type I interferons, and    Interferon-gamma.

-   221. The exogenous polynucleotide sequence of paragraph 220, wherein    the IL12 cytokine is an IL12p70 fusion protein.

-   222. The exogenous polynucleotide sequence of any one of paragraphs    210-221, wherein the chemokine is selected from the group consisting    of: CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-11 fusion, CCL19, CXCL9,    and XCL1.

-   223. The exogenous polynucleotide sequence of any one of paragraphs    210-222, wherein the growth factor is selected from the group    consisting of: Flt3L and GM-CSF.

-   224. The exogenous polynucleotide sequence of any one of paragraphs    210-223, wherein the co-activation molecule is selected from the    group consisting of: 4-1BBL and CD40L.

-   225. The exogenous polynucleotide sequence of any one of paragraphs    210-224, wherein the tumor microenvironment modifier is selected    from the group consisting of: adenosine deaminase, TGFbeta    inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and    HPGE2.

-   226. The exogenous polynucleotide sequence of paragraph 225, wherein    the TGFbeta inhibitors are selected from the group consisting of: an    anti-TGFbeta peptide, an anti-TGFbeta antibody, a TGFb-TRAP, and    combinations thereof

-   227. The exogenous polynucleotide sequence of paragraph 225, wherein    the immune checkpoint inhibitors comprise anti-PD-1 antibodies.

-   228. The exogenous polynucleotide sequence of paragraph 225, wherein    the VEGF inhibitors comprise anti-VEGF antibodies, anti-VEGF    peptides, or combinations thereof

-   229. The exogenous polynucleotide sequence of any one of paragraphs    181-225, wherein the first effector molecule and the second effector    molecule are human-derived effector molecules.

-   230. The exogenous polynucleotide sequence of any one of paragraphs    181-229, wherein the first effector molecule comprises IL12.

-   231. The exogenous polynucleotide sequence of any one of paragraphs    181-229, wherein the first effector molecule comprises an IL12p70    fusion protein.

-   232. The exogenous polynucleotide sequence of paragraph 231, wherein    the IL12p70 fusion protein is a human IL12p70 fusion protein.

-   233. The exogenous polynucleotide sequence of any one of paragraphs    230-232, wherein the second effector molecule comprises CCL21a.

-   234. The exogenous polynucleotide sequence of paragraph 233, wherein    the CCL21a is a human CCL21a.

-   235. The exogenous polynucleotide sequence of any one of paragraphs    230-232, wherein the second effector molecule comprises IL7.

-   236. The exogenous polynucleotide sequence of paragraph 235, wherein    the IL7 is a human IL7.

-   237. The exogenous polynucleotide sequence of any one of paragraphs    230-232, wherein the second effector molecule comprises IL21.

-   238. The exogenous polynucleotide sequence of paragraph 237, wherein    the IL21 is a human IL21.

-   239. The exogenous polynucleotide sequence of any one of paragraphs    181-238, wherein the expression cassette further comprises an E3    comprising a polynucleotide sequence encoding a third effector    molecule.

-   240. The exogenous polynucleotide sequence of paragraph 239, wherein    the third effector molecule comprises Flt3L.

-   241. The exogenous polynucleotide sequence of paragraph 239, wherein    the third effector molecule comprises anti-PD1.

-   242. The exogenous polynucleotide sequence of paragraph 241, wherein    the expression cassette further comprises an E4 comprising a    polynucleotide sequence encoding a fourth effector molecule.

-   243. The exogenous polynucleotide sequence of paragraph 242, wherein    the fourth effector molecule comprises adenosine deaminase.

-   244. The exogenous polynucleotide sequence of paragraph 239, wherein    the third effector molecule comprises adenosine deaminase.

-   245. The exogenous polynucleotide sequence of paragraph 239, wherein    the third effector molecule comprises CD40L.

-   246. The exogenous polynucleotide sequence of paragraph 239, wherein    the third effector molecule comprises a CXCL10-CXCL11 fusion    protein.

-   247. The exogenous polynucleotide sequence of paragraph 239, wherein    the third effector molecule comprises XCL1.

-   248. The exogenous polynucleotide sequence of paragraph 230, wherein    the second effector molecule comprises Flt3L.

-   249. The exogenous polynucleotide sequence of paragraph 230, wherein    the second effector molecule comprises a CXCL10-CXCL11 fusion    protein.

-   250. The exogenous polynucleotide sequence of paragraph 230, wherein    the second effector molecule comprises anti-PD1.

-   251. The exogenous polynucleotide sequence of paragraph 230, wherein    the second effector molecule comprises CD40L.

-   252. The exogenous polynucleotide sequence of any one of paragraphs    181-229, wherein the first effector molecule comprises    interferon-beta and the second effector molecule comprises Flt3L.

-   253. The exogenous polynucleotide sequence of any one of paragraphs    181-252, wherein the polynucleotide sequence encoding the first    effector molecule comprises a codon optimized polynucleotide    sequence.

-   254. The exogenous polynucleotide sequence of any one of paragraphs    181-253, wherein the polynucleotide sequence encoding the second    effector molecule comprises a codon optimized polynucleotide    sequence.

-   255. The exogenous polynucleotide sequence of any one of paragraphs    181-254, wherein the exogenous polynucleotide sequence comprises the    polynucleotide sequence shown in SEQ ID NO: 144.

-   256. An exogenous polynucleotide sequence comprising an SFFV    promoter and an expression cassette described in a formula, oriented    from 5′ to 3′, comprising    S1-E1-L-S2-E2    -   wherein    -   S1 comprises a polynucleotide sequence encoding a first signal        peptide, wherein the first signal peptide is a human IL12 signal        peptide;    -   E1 comprises a polynucleotide sequence encoding a first effector        molecule, wherein the first effector molecule is a human IL12p70        fusion protein;    -   L comprises a linker polynucleotide sequence, wherein the linker        polynucleotide sequence encodes Furin recognition polypeptide        sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome        skipping tag in a Furin:Gly-Ser-Gly:T2A orientation from        N-terminus to C-terminus;    -   S2 comprises a polynucleotide sequence encoding a second signal        peptide, wherein the second signal peptide is a human IL21        signal peptide;    -   E2 comprises a polynucleotide sequence encoding a second        effector molecule, wherein the second effector molecule is human        IL21; and    -   wherein the SFFV promoter is operably linked to the expression        cassette, the first signal peptide is operably linked to the        first effector molecule, and the second signal peptide is        operably linked to the second effector molecule.

-   257. The exogenous polynucleotide sequence of paragraph 256, wherein    the polynucleotide sequence comprises the polynucleotide sequence    shown in SEQ ID NO: 144.

-   258. An exogenous polynucleotide sequence comprising an SFFV    promoter and an expression cassette described in a formula, oriented    from 5′ to 3′, comprising    S1-E1-L-S2-E2    -   wherein    -   S1 comprises a polynucleotide sequence encoding a first signal        peptide, wherein the first signal peptide is a human IL12 signal        peptide;    -   E1 comprises a polynucleotide sequence encoding a first effector        molecule, wherein the first effector molecule is a human IL12p70        fusion protein;    -   L comprises a linker polynucleotide sequence, wherein the linker        polynucleotide sequence encodes Furin recognition polypeptide        sequence, a Gly-Ser-Gly polypeptide sequence, and a T2A ribosome        skipping tag in a Furin:Gly-Ser-Gly:T2A orientation from        N-terminus to C-terminus;    -   S2 comprises a polynucleotide sequence encoding a second signal        peptide, wherein the second signal peptide is a human IL21        signal peptide;    -   E2 comprises a polynucleotide sequence encoding a second        effector molecule, wherein the second effector molecule is human        IL21;    -   wherein the SFFV promoter is operably linked to the expression        cassette, the first signal peptide is operably linked to the        first effector molecule, and the second signal peptide is        operably linked to the second effector molecule;    -   wherein the promoter is operably linked to the expression        cassette such that the polynucleotides are capable of being        transcribed as a single polynucleotide comprising the formula        S1-E1-L-S2-E2; and    -   wherein the polynucleotide sequence comprises the polynucleotide        sequence shown in SEQ ID NO: 144.

-   259. The exogenous polynucleotide sequence of any one of paragraphs    181-258, wherein the exogenous polynucleotide sequence is encoded by    a nucleic acid selected from the group consisting of: a DNA, a cDNA,    an RNA, an mRNA, and a naked plasmid.

-   260. An expression vector comprising the exogenous polynucleotide    sequence of any one of paragraphs 181-259.

-   261. The expression vector of paragraph 260, wherein the expression    vector is a viral vector.

-   262. The expression vector of paragraph 261, wherein the viral    vector is a lentiviral vector.

-   263. A composition comprising the exogenous polynucleotide sequence    of any one of paragraphs 181-259, and a pharmaceutically acceptable    carrier.

-   264. An isolated cell comprising the exogenous polynucleotide    sequence of any one of paragraphs 181-259, the expression vector of    any one of paragraphs 260-262, or the composition of paragraph 263.

-   265. The isolated cell of paragraph 264, wherein the isolated cell    is selected from the group consisting of: a T cell, a CD8+ T cell, a    CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a    regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT)    cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating    lymphocyte (TIL), an innate lymphoid cell, a mast cell, an    eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage,    a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a    human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent    stem cell, an MSC, an induced pluripotent stem cell (iPSC), and an    iPSC-derived cell.

-   266. The isolated cell of paragraph 264, wherein the isolated cell    is an MSC.

-   267. The isolated cell of any one of paragraphs 264-266, wherein the    exogenous polynucleotide sequence is integrated into the genome of    the cell.

-   268. The isolated cell of any one of paragraphs 264-267, wherein the    exogenous polynucleotide sequence comprises one or more viral vector    polynucleotide sequences.

-   269. The isolated cell of paragraph 268, wherein the one or more    viral vector polynucleotide sequences comprise lentiviral,    retroviral, retrotransposon, or adenoviral polynucleotide sequences.

-   270. The isolated cell of paragraph 268, wherein the one or more    viral vector polynucleotide sequences comprise lentiviral    polynucleotide sequences.

-   271. The isolated cell of any one of paragraphs 264-270, wherein the    engineered cell is HLA-typed with reference to a subject in need of    therapeutic treatment.

-   272. The isolated cell of any one of paragraphs 264-271, wherein the    engineered cell is a human cell.

-   273. The isolated cell of paragraph 272, wherein the human cell is    an isolated cell from a subject.

-   274. The isolated cell of paragraph 273, wherein the isolated cell    is isolated from a tissue consisting of the group of: bone marrow,    adipose tissue, the umbilical cord, fetal liver, muscle, and lung    tissue.

-   275. The isolated cell of any one of paragraphs 264-272, wherein the    cell is a cultured cell.

-   276. The isolated cell of any one of paragraphs 264-275, wherein the    cell comprises a cellular marker phenotype comprising the cellular    markers CD105+, CD73+, and CD90+.

-   277. The isolated cell of paragraph 276, wherein the cellular marker    phenotype further comprises a phenotype lacking or substantially    lacking one or more cellular markers selected from the group    consisting of: CD45, CD34, CD14, CD11b, CD79α, CD19, HLA class II,    and combinations thereof

-   278. The isolated cell of any one of paragraphs 264-275, wherein the    cell comprises a cellular marker phenotype comprising CD105+, CD73+,    CD90+, CD45−, CD34−, CD14−; a cellular marker phenotype comprising    CD105+, CD73+, CD90+, CD11b−, CD79α−; a cellular marker phenotype    comprising CD105+, CD73+, CD90+, CD19−, HLA class II−; or a cellular    marker phenotype comprising CD73+, CD90+, CD105+, and CD166+,    CD11b−, CD14−, CD19−, CD34−, CD45−, and HLA−DR−.

-   279. The isolated cell of any one of paragraphs 264-278, wherein the    cellular marker phenotype further comprises a cellular marker    comprising a cognate receptor or a cognate receptor ligand for the    first effector molecule, the second effector molecule, or the first    and second effector molecules expressed in the cell.

-   280. The isolated cell of paragraph 279, wherein the receptor is    selected from the group consisting of: IL12RB1, IL12RB2, CCL7, and    combinations thereof

-   281. The isolated cell of any one of paragraphs 264-280, wherein the    cell secretes each effector molecule.

-   282. The isolated cell of paragraph 281, wherein the first effector    molecule is secreted at a ratio that is 10 fold higher relative to    secretion of the second effector molecule.

-   283. The isolated cell of any one of paragraphs 264-282, wherein the    cell further comprises an antigen recognizing receptor.

-   284. The isolated cell of paragraph 283, wherein the antigen    recognizing receptor comprises an antigen-binding domain.

-   285. The isolated cell of paragraph 284, wherein the antigen-binding    domain comprises an antibody, an antigen-binding fragment of an    antibody, a F(ab) fragment, a F(ab′) fragment, a single chain    variable fragment (scFv), or a single-domain antibody (sdAb).

-   286. The isolated cell of paragraph 284, wherein the antigen-binding    domain comprises a single chain variable fragment (scFv).

-   287. The isolated cell of paragraph 286, wherein the scFv comprises    a heavy chain variable domain (VH) and a light chain variable domain    (VL).

-   288. The isolated cell of paragraph 287, wherein the VH and VL are    separated by a peptide linker.

-   289. The isolated cell of paragraph 288, wherein the scFv comprises    the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain    variable domain, L is the peptide linker, and VL is the light chain    variable domain.

-   290. The isolated cell of any one of paragraphs 283-289, the antigen    recognizing receptor is a chimeric antigen receptor (CAR) or T cell    receptor (TCR).

-   291. The isolated cell of any one of paragraphs 283-289, the antigen    recognizing receptor is a chimeric antigen receptor (CAR).

-   292. The isolated cell of paragraph 291, wherein the CAR comprises    one or more intracellular signaling domains, and the one or more    intracellular signaling domains are selected from the group    consisting of: a CD3zeta-chain intracellular signaling domain, a    CD97 intracellular signaling domain, a CD11a-CD18 intracellular    signaling domain, a CD2 intracellular signaling domain, an ICOS    intracellular signaling domain, a CD27 intracellular signaling    domain, a CD154 intracellular signaling domain, a CD8 intracellular    signaling domain, an OX40 intracellular signaling domain, a 4-1BB    intracellular signaling domain, a CD28 intracellular signaling    domain, a ZAP40 intracellular signaling domain, a CD30 intracellular    signaling domain, a GITR intracellular signaling domain, an HVEM    intracellular signaling domain, a DAP10 intracellular signaling    domain, a DAP12 intracellular signaling domain, and a MyD88    intracellular signaling domain.

-   293. The isolated cell of paragraph 291 or paragraph 292, wherein    the CAR comprises a transmembrane domain, and the transmembrane    domain is selected from the group consisting of: a CD8 transmembrane    domain, a CD28 transmembrane domain a CD3zeta-chain transmembrane    domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an    OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4    transmembrane domain, a PD-1 transmembrane domain, a LAG-3    transmembrane domain, a 2B4 transmembrane domain, and a BTLA    transmembrane domain.

-   294. The isolated cell of any one of paragraphs 291-293, wherein the    CAR comprises a spacer region between the antigen-binding domain and    the transmembrane domain.

-   295. A virus comprising the exogenous polynucleotide sequence of any    one of paragraphs 181-259 or the expression vector of any one of    paragraphs 260-262.

-   296. The virus of paragraph 295, wherein the virus is selected from    the group consisting of: a lentivirus, a retrovirus, a    retrotransposon, and an adenovirus.

-   297. The virus of paragraph 295, wherein the virus is a lentivirus.

-   298. A method of reducing tumor volume in a subject, the method    comprising delivering to a subject having a tumor a composition    comprising cells engineered to produce multiple effector molecules    that modulate tumor-mediated immunosuppressive mechanisms, in an    effective amount to reduce the volume of the tumor, wherein the    engineered cells comprise:

a) a promoter; and

b) an exogenous polynucleotide sequence comprising an expressioncassette described in a formula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2

-   -   -   wherein        -   S1 comprises a polynucleotide sequence encoding a first            signal peptide,        -   E1 comprises a polynucleotide sequence encoding a first            effector molecule,        -   L comprises a linker polynucleotide sequence,        -   S2 comprises a polynucleotide sequence encoding a second            signal peptide,        -   E2 comprises a polynucleotide sequence encoding a second            effector molecule, and        -   wherein the promoter is operably linked to the expression            cassette, the first signal peptide is operably linked to the            first effector molecule, and the second signal peptide is            operably linked to the second effector molecule, and        -   wherein the engineered cell is selected from the group            consisting of: a mesenchymal stem cell (MSC), stem cell,            immune cell, natural killer (NK) cell, NKT cell, innate            lymphoid cell, tumor-infiltrating lymphocyte (TIL), mast            cell, eosinophil, basophil, monocyte, macrophage,            neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T            cell, CD4+ T cell, cytotoxic T lymphocyte (CTL),            viral-specific T cell, gamma-delta T cell, T regulatory            cell, and B cell.

-   299. A method of reducing tumor volume in a subject, the method    comprising delivering to a subject having a tumor a composition    comprising cells engineered to produce IL12 and IL21, in an    effective amount to reduce the volume of the tumor, wherein the    engineered cells comprise a construct, wherein the construct    comprises:    -   a) an SFFV promoter; and    -   b) an exogenous polynucleotide sequence comprising an expression        cassette described in a formula, oriented from 5′ to 3′,        comprising        S1-E1-L-S2-E2        -   wherein        -   S1 comprises a polynucleotide sequence encoding a first            signal peptide, wherein the first signal peptide is a human            IL12 signal peptide;        -   E1 comprises a polynucleotide sequence encoding a first            effector molecule, wherein the first effector molecule is a            human IL12p70 fusion protein;        -   L comprises a linker polynucleotide sequence, wherein the            linker polynucleotide sequence encodes Furin recognition            polypeptide sequence, a Gly-Ser-Gly polypeptide sequence,            and a T2A ribosome skipping tag in a Furin:Gly-Ser-Gly:T2A            orientation from N-terminus to C-terminus;        -   S2 comprises a polynucleotide sequence encoding a second            signal peptide, wherein the second signal peptide is a human            IL21 signal peptide;        -   E2 comprises a polynucleotide sequence encoding a second            effector molecule, wherein the second effector molecule is            human IL21; and        -   wherein the SFFV promoter is operably linked to the            expression cassette, the first signal peptide is operably            linked to the first effector molecule, and the second signal            peptide is operably linked to the second effector molecule,            and        -   wherein the engineered cell is selected from the group            consisting of: a mesenchymal stem cell (MSC), stem cell,            immune cell, natural killer (NK) cell, NKT cell, innate            lymphoid cell, tumor-infiltrating lymphocyte (TIL), mast            cell, eosinophil, basophil, monocyte, macrophage,            neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T            cell, CD4+ T cell, cytotoxic T lymphocyte (CTL),            viral-specific T cell, gamma-delta T cell, T regulatory            cell, and B cell.

-   300. The method of paragraph 299, wherein the construct comprises    the polynucleotide sequence shown in SEQ ID NO: 144.

-   301. The method of any one of paragraphs 298-300, wherein the method    further comprises administering a checkpoint inhibitor.

-   302. The method of paragraph 301, wherein the checkpoint inhibitor    is an anti-PD-1 antibody, anti-PD-1L antibody or an anti-CTLA-4    antibody.

-   303. The method of any one of paragraphs 298-302, wherein the method    further comprises administering an anti-CD40 antibody.

-   304. The method of any one of paragraphs 298-303, wherein the tumor    is selected from the group consisting of: an adenocarcinoma, an    acute myeloid leukemia (AML), an acute lymphoblastic B-cell leukemia    (BALL), an acute lymphoblastic T-cell leukemia (TALL), a B-cell    prolymphocytic leukemia, a bladder tumor, a brain tumor, a breast    tumor, a cervical tumor, a chronic lymphocytic leukemia, a chronic    myeloid leukemia (CML), a colorectal tumor, an esophageal tumor, a    glioma, a kidney tumor, a liver tumor, a lung tumor, a lymphoma, a    melanoma, a mesothelioma, a myelodysplasia, an ovarian tumor, a    pancreatic tumor, a plasma cell myeloma, a prostate tumor, a skin    tumor, a thyroid tumor, and a uterine tumor.

-   305. The method of any one of paragraphs 298-303, wherein the tumor    is an ovarian tumor.

-   306. The method of any one of paragraphs 298-303, wherein the tumor    is a tumor located in a peritoneal space.

-   307. The method of any one of paragraphs 298-306, wherein the    administering comprises systemic administration, intraperitoneal    administration, or intratumoral administration

-   308. The method of any one of paragraphs 298-307, wherein the volume    of the tumor is reduced by at least 25% relative to a control,    optionally wherein the control is an unmodified cell.

-   309. The method of paragraph 307, wherein the volume of the tumor is    reduced by at least 50% relative to a control, optionally wherein    the control is an unmodified cell.

-   310. The method of paragraph 309, wherein the volume of the tumor is    reduced by at least 75% relative to a control, optionally wherein    the control is an unmodified cell.

-   311. A method of reducing tumor volume in a subject, the method    comprising delivering to a subject having a tumor a composition    capable of engineering an cell to produce multiple effector    molecules that modulate tumor-mediated immunosuppressive mechanisms,    in an effective amount to reduce the volume of the tumor, wherein    each engineered cell comprises:    -   a) a promoter; and    -   b) an exogenous polynucleotide sequence comprising an expression        cassette described in a formula, oriented from 5′ to 3′,        comprising        S1-E1-L-S2-E2        -   wherein        -   S1 comprises a polynucleotide sequence encoding a first            signal peptide,        -   E1 comprises a polynucleotide sequence encoding a first            effector molecule,        -   L comprises a linker polynucleotide sequence,        -   S2 comprises a polynucleotide sequence encoding a second            signal peptide,        -   E2 comprises a polynucleotide sequence encoding a second            effector molecule, and        -   wherein the promoter is operably linked to the expression            cassette, the first signal peptide is operably linked to the            first effector molecule, and the second signal peptide is            operably linked to the second effector molecule, and        -   wherein the engineered cell is selected from the group            consisting of: a mesenchymal stem cell (MSC), stem cell,            immune cell, natural killer (NK) cell, NKT cell, innate            lymphoid cell, tumor-infiltrating lymphocyte (TIL), mast            cell, eosinophil, basophil, monocyte, macrophage,            neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T            cell, CD4+ T cell, cytotoxic T lymphocyte (CTL),            viral-specific T cell, gamma-delta T cell, T regulatory            cell, and B cell.

-   312. A method of reducing tumor volume in a subject, the method    comprising delivering to a subject having a tumor a composition    capable of engineering a cell to produce IL12 and IL21, in an    effective amount to reduce the volume of the tumor, wherein the    engineered cell comprises a construct, wherein the construct    comprises:    -   a) an SFFV promoter; and    -   b) an exogenous polynucleotide sequence comprising an expression        cassette described in a formula, oriented from 5′ to 3′,        comprising        S1-E1-L-S2-E2        -   wherein        -   S1 comprises a polynucleotide sequence encoding a first            signal peptide, wherein the first signal peptide is a human            IL12 signal peptide;        -   E1 comprises a polynucleotide sequence encoding a first            effector molecule, wherein the first effector molecule is a            human IL12p70 fusion protein;        -   L comprises a linker polynucleotide sequence, wherein the            linker polynucleotide sequence encodes Furin recognition            polypeptide sequence, a Gly-Ser-Gly polypeptide sequence,            and a T2A ribosome skipping tag in a Furin:Gly-Ser-Gly:T2A            orientation from N-terminus to C-terminus;        -   S2 comprises a polynucleotide sequence encoding a second            signal peptide, wherein the second signal peptide is a human            IL21 signal peptide;        -   E2 comprises a polynucleotide sequence encoding a second            effector molecule, wherein the second effector molecule is            human IL21; and        -   wherein the SFFV promoter is operably linked to the            expression cassette, the first signal peptide is operably            linked to the first effector molecule, and the second signal            peptide is operably linked to the second effector molecule,            and        -   wherein the engineered cell is selected from the group            consisting of: a mesenchymal stem cell (MSC), stem cell,            immune cell, natural killer (NK) cell, NKT cell, innate            lymphoid cell, tumor-infiltrating lymphocyte (TIL), mast            cell, eosinophil, basophil, monocyte, macrophage,            neutrophil, myeloid cell, dendritic cell, T cell, CD8+ T            cell, CD4+ T cell, cytotoxic T lymphocyte (CTL),            viral-specific T cell, gamma-delta T cell, T regulatory            cell, and B cell.

-   313. The method of paragraph 312, wherein the construct comprises    the polynucleotide sequence shown in SEQ ID NO: 144.

-   314. The method of any one of paragraphs 311-313, wherein the    composition comprises a delivery system selected from the group    consisting of: a viral system, a transposon system, and a nuclease    genomic editing system.

-   315. The method of paragraph 314, wherein the viral system is    selected from the group consisting of: a lentivirus, a retrovirus, a    retrotransposon, and an adenovirus.

-   316. The method of paragraph 314, wherein the nuclease genomic    editing system is selected from the group consisting of: a    zinc-finger system, a TALEN system, and a CRISPR system.

-   317. The method of any one of paragraphs 311-316, wherein the tumor    is selected from the group consisting of: an adenocarcinoma, an    acute myeloid leukemia (AML), an acute lymphoblastic B-cell leukemia    (BALL), an acute lymphoblastic T-cell leukemia (TALL), a B-cell    prolymphocytic leukemia, a bladder tumor, a brain tumor, a breast    tumor, a cervical tumor, a chronic lymphocytic leukemia, a chronic    myeloid leukemia (CML), a colorectal tumor, an esophageal tumor, a    glioma, a kidney tumor, a liver tumor, a lung tumor, a lymphoma, a    melanoma, a mesothelioma, a myelodysplasia, an ovarian tumor, a    pancreatic tumor, a plasma cell myeloma, a prostate tumor, a skin    tumor, a thyroid tumor, and a uterine tumor.

-   318. The method of any one of paragraphs 311-317, wherein the    administering comprises systemic administration, intraperitoneal    administration, or intratumoral administration.

EXAMPLES Example 1

This Example describes the in vitro characterization of MSCs withindividual and combination immunotherapy payloads. Direct anti-cancereffects of immunotherapy-expressing MSCs on cancer cells are firstmeasured. Next, the effects of immunotherapy-expressing MSCs onco-cultures with primary immune cells (focusing on T cells) and cancercells are measured. The immuno-stimulatory properties ofimmunotherapy-expressing MSCs are rank-ordered based on inflammatorybiomarker panels in both mouse and human cell systems.Immunotherapy-expressing MSCs that significantly enhance cancer cellkilling either on their own or together with T cells are identified, andthe top candidates to advance to in vivo testing are selected.

Methods: Immunotherapy-expressing MSCs are engineered to express theeffector molecules listed in Table 1 are evaluated for their functionaleffects using in vitro models relevant to cancer therapy. Human ovariancancer cells (e.g., OVCAR8 and SKOV3) and human immune cells isolatedfrom circulating PBMCs are used to test the hMSCs expressing hITs. Mouseovarian cancer cells (e.g., ID8) and mouse immune cells are used to testthe mMSCs expressing mITs.

Checkpoint inhibitors. Cell-binding assays are used to verify theactivity of the expressed antibodies. The targets of the antibodies,CTLA4 and PD1, both negatively regulate T cells, but they areupregulated at different stages of T-cell activation (Boutros C, et al.(2016) Nat Rev Clin Oncol 13(8):473-486; Valsecchi M E (2015) New Engl JMed 373(13):1270-1270). CTLA4 is briefly upregulated in the primingphase, whereas PD1 is consistently expressed in the effector phase of Tcell activation (Pardoll D M (2012) Nat Rev Cancer 12(4):252-264; LegatA, et al. (2013) Front Immunol 4:455). Anti-CTLA4 antibody binds toCTLA4 on the T-cell surface, blocking CTLA4 from shutting down T-cellactivation in the early stage, and the human anti-PD1 antibody binds toPD1, preventing tumor cells from inhibiting T-cell activity.

T cells are isolated from PBMC by negative selection using EASYSEP™magnetic bead (STEMCELL Technologies). The isolated T cells areactivated by Human T-Activator CD3/28 Dynabeads (Thermo Fisher) andexpression of CTLA-4 and PD-1 is monitored over 5 days to select foroptimal timing of expression for each surface marker. On the appropriatedays, conditioned media from the MSCs expressing antibodies for CTLA-4or PD-1, or control conditioned media from non-expressing MSCs, areapplied to the activated T cells to validate directcell-surface-receptor binding of these antibodies. Fluorochrome-labeledsecondary detection antibodies together with flow cytometry shouldconfirm binding.

Chemokines. CCL21 chemokine functionality is confirmed using cellmigration assays and isolated naïve T cells, which express chemokinereceptor CCR7 that is responsive to CCL21 chemotaxis. Specifically,CCL21-expressing or control MSCs are added to one compartment of atrans-well and then cell migration is assessed by isolated naïve T cellsfrom the other compartment, followed by enumeration of numbers ofmigrated T cells (Justus C R, et al. (2014) J Vis Exp (88)).

Cytokines. The activity of IL2, IL12, and IL15 is measured. ELISA assaysspecific to IL2, IL12, and IL15 are used to detect levels of thesecytokines in MSC supernatants. Functional bioactivity assays employ theCTLL-2 cell line to assess of IL2 or IL15-mediated proliferation, or theNKG cell line to assess IL12-mediated IFN-gamma production by MSCsupernatants. Multiplexed cytokine profiling assays using LUMINEX®technology may also be used to assess cytokine expression and effects onimmune cells.

STING pathway. STING pathway activation is measured with theconstitutive STING mutant payload. Using LUMINEX® beads, the secretionof Type I interferons (e.g. IFN-alpha2 and IFN-beta) with expression ofthe STING mutant are profiled in MSCs.

Direct effects of immunotherapy-expressing MSCs on ovarian cancer cells.Any direct effects of MSCs on ovarian cancer cell growth and viabilityare tested in vitro. For example, mMSC or hMSC candidates areco-cultured with the mouse ovarian cancer cell line (ID8) or humanovarian cancer cell lines (OVCAR8 and SKOV3) and cancer cellcytotoxicity is measured by the well-characterized lactate dehydrogenase(LDH) assay. After 24 hours of co-culture, the supernatants arecollected and measured for LDH levels correlated to cellular death viaan enzymatic reaction that is subsequently quantified by specificabsorbance on a plate reader. Additionally, cancer cell numbers areassessed by counting live versus dead cells by Trypan Blue exclusion andlive versus apoptotic/dead cells by flow cytometric measurement usingAnnexin-V and propidium iodide staining.

Effects of immunotherapy-expressing MSCs on T cell and ovarian cancercell co-culture systems. Tests determine whetherimmunotherapy-expressing MSCs can stimulate immune cells, such as Tcells, to have improved anti-cancer activity against ovarian cancercells in vitro. Specifically, mMSC-mIT candidates are co-cultured withmouse splenocytes and the ID8 cancer cell line, or hMSC-hIT candidatesare co-cultured with human PBMCs and the OVCAR8 or SKOV3 cell lines. Theco-culture assays entail using PBMCs/splenocytes with the ovarian cancercells, with or without the MSCs, and stimulation with anti-CD3/28 beads.To assess cancer cell death, 16 hour killing assays are performed usingtechniques such as LDH cytotoxicity measurements, combining dye-labeledovarian cancer cells with non-labeled effector PBMCs/splenocytes atfixed ratios and assaying killing by flow cytometry (Jedema I, et al.(2004) Blood 103(7):2677-2682), and apoptosis readouts by flow cytometryusing Annexin-V with propidium iodide. T cell activation/proliferationis specifically assay by CFSE cell division at 3-5 days and cytokineproduction of IFN-gamma at 1-3 days.

An alternative strategy to generate T cells expressing CTLA-4 and PD1 isto activate with phytohaemagglutinin (PHA) to express the cell surfacereceptors PD1 and CTLA4. On Day 3, ˜99% of the activated T cells shouldexpress PD1 while ˜15% of them should express CTLA4 (Pardoll D M (2012)Nat Rev Cancer 12(4):252-264; Legat A, et al. (2013) Front Immunol4:455). On Day 10, the activated T cells should be in the effectorphase, when CTLA4 expression is downregulated but PD1 expression ismaintained. Direct cell-surface-receptor binding of these antibodies isevaluated. On Day 3 and Day 10 post-induction, MSCs with the respectivecheckpoint inhibitor antibody expression constructs are applied to the Tcell cultures. Labeled detection antibodies are used together with flowcytometry to confirm binding. Commercial antibodies are used ascontrols.

Example 2

This Example describes the in vivo characterization of MSCs expressingimmunotherapy payloads in a syngeneic ovarian cancer model. Theanti-tumor efficacy of immunotherapy-expressing MSCs is characterizedusing syngeneic mouse models of ovarian cancer (mMSC-mIT with mouseimmune system). Tumor homing of engineered MSCs and expression ofindividual and combinatorial immunotherapies in a syngeneic ovarianmouse model are measured. Ovarian tumor burden and mouse survival withengineered MSC treatments are also measured. This Example shoulddemonstrate selective homing of engineered MSCs to the TME and localizedproduction of immunotherapy factors in ovarian tumors versus other bodysites. This Example should also demonstrate significant reductions intumor burden and extension of mouse survival withimmunotherapy-expressing engineered MSCs.

Methods: The mouse ID8 cell line originated from spontaneoustransformation of mouse ovarian epithelial surface cells (MOSE), is usedto create a syngeneic ovarian tumor model (Roby K F, et al. (2000)Carcinogenesis 21(4):585-591). Derivatives of the ID8 cell line are alsoused (e.g., ID8-VEGF (ID8-Defb29/Vegf-a), ID8-P53DN, ID8-P53KO-PTEN KO,ID8-P53KO-BRCA2 KO, ID8-P53KO-BRCA1 KO, ID8-PD53KO-Nf1KO). The ID8 cellline is infected with a lentivirus expressing Renilla luciferase (rLuc)to allow for in vivo bioluminescence imaging that is orthogonal to MSCsexpressing Firefly luciferase (ffLuc). Successful rLuc expression isconfirmed in ID8 in vitro prior to establishing the syngeneic ovariancancer model in mice. For the syngeneic model, 5×10⁵ ID8 cells areinjected into the peritoneal cavity of C57BL/6 mice between 6 to 8 weeksold (36, 54). MSCs are engineered as in Example 1, along with anffLuc-expressing plasmid.

mMSC-mIT candidates are introduced into the syngeneic mouse modelstarting on day 25 (after tumor cell injection) at a dose of 10⁶ MSC peranimal once per week for 5 weeks (Dembinski J L, et al. (2013)Cytotherapy 15(1):20-32). The ovarian tumor load and mMSC-mIT candidatesare visualized over time through rLuc and ffLuc bioluminescence imaging,respectively, as well as histological analyses following terminal timepoints. Mice are euthanized when they develop signs of distress, such asbody-weight loss, ruffled fur, poor body posture, distended abdomen, andjaundice. Survival curves for the mice are measured. Distal metastasisof tumor cells is quantified by bioluminescence imaging (BLI) and bynecropsy at time of euthanasia. Immune system profiling and activity ismeasured at different time points as biomarkers of response to thetherapy.

To assess for variability in the expected anti-tumor effects of theMSCs, the dose of ID8 cells used to establish the model is varied (e.g.,increase the number of cells to 5×10⁶), the dose of MSCs used ischanged, and the time when MSCs are delivered after tumor establishmentis modulated.

Even though mMSCs have been shown to home to ovarian tumors in mousemodels, it is possible that some payloads disrupt this homing activity.In these instances, expression of these payloads may be engineered to beinducible. This can be achieved, for example, with a phloretin-induciblesystem (Gitzinger M, et al. (2009) Proc Natl Acad Sci USA106(26):10638-10643). Alternatively, the Dimerizer system may be used tolink a synthetic zinc-finger DNA-binding domain with a transactivatordomain using a small molecule (Clackson T, et al. (1998) Proc Natl AcadSci USA 95(18):10437-10442). Alternatively or additionally, induciblepayload expression constructs that are triggered in the tumormicroenvironment based on signals such as low O₂ may be constructed.

Lentiviral ffLuc constructs may also be used to infect MSCs.

Example 3

This Example describes the in vivo characterization of the efficacy ofMSCs expressing immunotherapy payloads in xenograft models of humanovarian cancer in mice with human immune cells. The activity ofengineered MSCs in human ovarian cancer models in immunodeficient micethat are engrafted with human immune cells via CD34+ cell transplants(hMSC-hIT with humanized immune system) is tested. Homing of engineeredMSCs and expression of individual and combinatorial immunotherapies inhuman xenograft ovarian tumors in mice with human immune cells aremeasured. Ovarian tumor burden and mouse survival with engineered MSCtreatments are also tested. This Example should demonstrate elevatedhoming of engineered MSCs and localized production of immunotherapyfactors into human xenograft ovarian tumors versus other body sites inmice. This Example should also demonstrate significant reductions intumor burden and extension of mouse survival withimmunotherapy-expressing engineered MSCs correlating with changes in theimmune system composition.

Methods. To enable translation of engineered MSCs into human clinicaltrials, hMSC-hIT constructs are tested in humanized mouse models ofhuman cancers. The effects of the immunotherapy-expressing hMSCs in miceare modeled by using xenografts of human ovarian cancer cell lines inimmuno-deficient mice (NSG) engrafted with CD34⁺ hematopoietic stemcells (HSCs).

For human ovarian cancer cells, OVCAR8 and SKOV3 cell lines are used.Similar assays as described in Example 3 are used to investigate tumorload and mouse survival over time.

Two alternative approaches may also be used. (1) Human T cells can beinfused into the mice. (2) Human PBMCs can be infused into the mice.

Expression Vector: pL + MCS (SEQ ID NO: 111)ACGCGTGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGACGAACCACTGAATTGCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCTAGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCACTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGAATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATCAAAATTTTATCTCGACATGGTGGCGACCGGTAGCGCTAGCGGATCGATAAGCTTGATATCGCCTGCAGCCGAATTCCTTGACTTGGGATCCGCGTCAAGTGGAGCAAGGCAGGTGGACAGTCCTGCAGGCATGCGTGACTGACTGAGGCCGCGACTCTAGTTTAAACTGCGTGACTGACTCTAGAAGATCCGGCAGTGCGGCCGCGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAAATAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCATCTTATTATTCAGTATTTATAACTTGCAAAGAAATGAATATCAGAGAGTGAGAGGAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGCTCTAGCTATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGACTTTTGCAGAGACGGCCCAAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTG

Example 4: 4T1 Triple Negative Breast Carcinoma

In the following experiments, MSCs were engineered to express one of thefollowing effector molecules, then administered, alone or incombinations, to an orthotopic breast cancer mouse model: IFNβ, IFNγ,IL12, IL15, IL36γ, IL7, TRAIL, cGAS, CCL21a, OX40L, CD40L, or HACv-PD1.In some examples, a checkpoint inhibitor (anti-CD40, anti-PD1, oranti-CTLA-4 antibody) was injected in combination with administrationwith the engineered MSCs.

MSC Homing

The following experiments demonstrate that murine MSCs home to tumors inan orthotopic mouse model of breast cancer. Luciferase-expressing 4T1breast tumor cells (5×10⁵) were orthotopically implanted into the dorsalfat pad of female BALB/cJ mice. After 5 days, mice wereintraperitoneally injected with 1 million fluorescently-labeled (withXenoLight DiR (Caliper Life Sciences)) murine BM-derived MSCs (BM-MSCs,therapeutic cells). At days 1 and 7 after MSC injection, fluorescenceanalysis was used to determine MSC localization using the Ami HT liveanimal imager (Spectral Instruments). On day 7, tumor localization andsize was determined through the 4T1 cell's luciferase bioluminescencereporter using the Ami HT imager. As shown in FIG. 3, the injected MSCsco-localized to the site of the tumor, indicating that these cells do infact specifically home in vivo to sites of 4T1 breast tumors. Theinjected MSCs home to tumors within one day and persist for over 7 days.In contrast, injected MSCs do not home to the dorsum in the absence oftumor in normal mice. These results suggest that MSCs can be used as adelivery vehicle for anti-cancer molecules, proteins or compounds.

To determine whether engineered human MSCs can home toward mouse tumors,different lines of engineered human MSC expressing either GFP, IL2 orCCL21a were injected into BALB/c mice with 4T1 tumors. Efficacy wasdetermined by tumor volume from caliper measurement every other day.FIGS. 11A-11B show that human MSCs do not home to mouse 4T1 tumors.

In Vivo Efficacy

The following experiments demonstrate the in vivo efficacy of MSCsexpressing immunotherapy effectors (payloads) in the orthotopic model ofbreast cancer. 4T1-Neo-Fluc mouse breast tumor cells (Imanis LifeSciences, 5×10⁵ cells) were implanted orthotopically into the dorsal fatpad of female BALB/cJ mice (The Jackson Laboratory). Mice were thenrandomized into the treatment groups 5 days after tumor implantation.Mice received intraperitoneal injection of either control MSC growthmedia or engineered MSCs (2×10⁶ cells) expressing differentimmunotherapy effectors (payloads) once a week for two weeks. Eachimmunotherapy was expressed by a different MSC, and MSCs were combined(1:1 ratio) for combinatorial treatment. Tumor growth was monitored bycaliper measurements every other day, and mouse weights were recordedtwice weekly. Mice were euthanized 14 days after first MSC treatment andtissues were collected for further analysis.

FIG. 4 shows that tumor growth was delayed in mice treated withengineered MSCs expressed combinatorial genes IL-12 and CCL21a comparedto controls treated with media.

FIGS. 5A-5C show that engineered MSCs that express single immunotherapyeffectors (e.g., IFN-β, IFN-γ, IL-12 or CCL21a) inhibited growth ofsyngeneic 4T1 mouse tumors compared to media-treated mice. Surprisingly,a synergistic effect on tumor growth was observed when the immunotherapyeffectors were combined, particularly the combination of IL-12 andCCL21a, and the combination of IFN-β, IFN-γ, IL-12 and CCL21a (FIGS.5A-5C).

FIGS. 6A-6B show that engineered MSCs expressing OX40L, TRAIL, IL15,cGAS, or combinations thereof do not inhibit tumor growth.

FIGS. 7A-7B show that engineered MSCs expressing IL-12 and CCL21ainhibit tumor growth; however the addition of anti-CD40 antibody doesnot reduce tumor growth.

FIGS. 8A-8B show that engineered MSCs expressing OX40L, TRAIL, IL15,HACvPD-1, or combinations thereof do not inhibit tumor growthsignificantly in a subcutaneous breast cancer model.

FIGS. 9A-9B show that engineered MSCs expressing IL-12 and CCL21ainhibit tumor growth; however the combination of MSCs expressing CCL21a,IL-36 gamma and IL-7 does not reduce tumor growth. Some of the effectorcombinations tested, however, may cause toxicity.

Dose Escalation

A dose escalation study was performed. This experiment determined thatengineered MSC cell expression GFP does not elicit toxicity (FIGS.10A-10B).

Effect on Large Tumors

This experiment tested whether engineered mouse MSCs expressing IL12 andCCL21a can reduce tumor burden from larger tumor (>800 mm³). Largertumor are more difficult to treat than small tumor, and this experimentdemonstrates this effector combination can reduce tumor expansion (FIGS.12A-12B).

Checkpoint Inhibitors

FIG. 13A shows that engineered MSCs expressing IL-12 and CCL21 aresufficient to inhibit tumor growth, although the addition of acheckpoint inhibitor (anti-PD-1 antibody or anti-CTLA-4 antibody) byinjection did not increase efficacy in a subcutaneous tumor model.

Example 5: CT26 Colorectal Carcinoma

In the following experiments, MSCs were engineered to express one of thefollowing effector molecules, then administered, alone or incombinations, to a colorectal carcinoma mouse model: IFNβ, IL12, IL15,IL36γ, IL7, CCL21a, HACv-PD1, or 41BB. In some examples, a checkpointinhibitor (anti-CD40 or anti-CTLA-4 antibody) was injected incombination with administration with the engineered MSCs.

FIG. 14 shows that engineered MSCs expressing IL-12 and CCL21a inducedsignificant tumor growth delay.

FIG. 15 shows tumor growth kinetics in the CT26 mouse model to determineoptimal time for dosing the engineered MSC cells.

In Vivo Efficacy

The following experiments demonstrate the in vivo efficacy of MSCsexpressing immunotherapy effectors (payloads) in the subcutaneous mousemodel of colon (colorectal) cancer. CT26-Neo-Fluc mouse colon cancercells (Imanis Life Sciences, 5×10⁵) were injected subcutaneously intothe flanks of female BALB/cJ mice (The Jackson Laboratory). Seven daysafter tumor implantation, mice were then randomized into the followingtreatment groups: control MSC growth media, engineered MSCs(MSC-12+CCL21a), anti-CD40 antibody, anti-CTLA4 antibody (Bio X cell),MSC-12+CCL21a in combination with anti-CD40 antibody or MSC-12+CCL21a incombination with anti-CTLA4 antibody. Engineered MSCs (2×10⁶ cells) wereinjected intraperitoneally (ip) once a week for two weeks (Day 0 and 7).Anti-CD40 antibodies were injected ip (100 μg) on Days 0 and 3.Anti-CTLA4 antibodies were injected ip (100 μg) on Days 0, 3 and 7.Tumor growth was monitored by caliper measurements every other day, andmouse weights were recorded twice weekly. Mice were euthanized 11 daysafter first MSC treatment and tumors were collected and weighed. Thetumor weight of individual mice in each treatment group was measured andthe results are shown in the bottom left of FIG. 16B (left graph). Theaverage tumor volume of each treatment group was monitored over time(FIG. 16B, right graph). Treatment Groups 2 (IL-12+CCL21a+ anti-CTLA4antibody), 4 (IL-12+CCL21a) and 7 (IL-12+CCL21a+ anti-CD40 antibody)inhibited the average growth of CT26 colon tumors compared toGFP-treated mice (FIG. 16B, right graph). Similar results were observedwhen the tumor volume of individual mice in each treatment group wasmeasured over time (FIG. 16A). Therefore, combinatorial treatment withMSCs expressing immunotherapies inhibited the growth of colon cancercells in vivo.

FIG. 18A shows that engineered MSCs expressing IL-12, CCL21a, and eitherIL15 or HACvPD-1 inhibit tumor growth significantly in a mouse model ofcolorectal cancer. FIG. 18B shows the tumor weight for individual micein each treatment. FIG. 18C is a representative graph of theinfiltrating immune population within the tumor microenvironment. FIG.18D shows the percentage of regulatory T cells (Treg) in the total CD3population. There was a significant decrease in the numbers of Tregs inthe tumor microenvironment treated with engineered MSC-IL2 and CCL21a.FIG. 18E correlates the percentage of immune infiltration with tumorweight. Samples with increase in lymphocytes (CD3+) were found tocorrelate with low tumor weight, while samples with high myeloid(CD11b+) infiltration were correlated with higher tumor burden.

Long-Term Survival

Mice were dosed twice with different concentration of engineeredMSC-IL12 and CCL21a therapy in combination with injected anti-CD40antibody. After the second dose, tumor volume was monitored twice a weekuntil tumor burden is greater than 1500 mm³ and the mice weresacrificed. FIG. 17A shows the tumor volume of the individual group.FIG. 17B, left graph, tracks the mice weight and tumor volume fromindividual group over time. FIG. 17B, right graph, shows the survivalplot of the different groups.

MSC Efficacy

FIG. 20A shows the tumor volume for individual mice in each treatment.FIG. 20B shows the tumor weight for individual mice in each treatment.Efficacy was determined by tumor volume from caliper measurement everyother day.

Tumor Growth Kinetics

FIGS. 21A-21B show the kinetics of CT26-LUC (luciferase) tumor growth inthe intraperitoneal space. A CT26 cell line was injected at day 0 andthree (3) mice were harvested at day 7, day 10, day 14, and day 18 todetermine the kinetics of tumor growth. The first row of FIG. 21Ameasures the mice body weight and ROI with an IVIS imager to monitortumor burden. The second row monitors the tumor weight and the ROI ofthe tumor of individual mice in each group. The third row correlates thetumor weight with either whole body ROI or tumor ROI. FIG. 21B shows theimmune profile of three (3) mice in the day 18 group to betterunderstand the tumor microenvironment.

Tumor Infiltrate Statistics/Immune Percentage/Tumor Weight

Subcutaneous Mouse Model

FIG. 22A includes data indicating that engineered MSCs expressing IL-12and CCL21a inhibit tumor growth in an subcutaneous mouse model ofcolorectal cancer; however the combination of MSCs expressing CCL21a andIL-36 gamma or IL-7 does not reduce tumor growth. FIGS. 23A-23B includethe tumor immune infiltrate statistics. Three mice were selected fromPBS, Naïve MSC, and MSC-IL12+MSC-CCL21a (combo) group to run flowcytometry to immune profile tumor microenvironment. FIG. 23A shows asignificant increase in infiltrating CD3 and CD8 cytotoxic T populationin the combo group compared to the group dosed with naïve MSC. FIG. 23Bshows a significant reduction in granulocytic myeloid-derived suppressorcells (gMDSCs) and macrophage population in the combo group compared togroup treated with Naïve MSC.

FIGS. 24A-24B include data relating to immune percentage and tumorweight, showing that samples with more CD3+ and CD8+ T cells (top leftand center graph) correlate strongly with a decrease in tumor weight.These figures also show that samples with fewer CD11b myeloid cells,including macrophage, dendritic cells, and MDSC, display lower tumorburden (lower center and right graph of FIG. 24A and upper row of FIG.24B).

Orthotopic Mouse Model

FIG. 26A shows that engineered MSCs expressing IL-12 and CCL21a, orCCL21a and IFN-β, inhibit tumor growth in an orthotopic mouse model ofcolorectal cancer; however the combination of MSCs expressing CCL21a ands41BBL does not reduce tumor growth. Each effector was expressed by adifferent MSC, and the MSCs were combined (at a 1:1 ratio) forcombinatorial treatment. Each chart shows the effect of engineered MSCsexpressing the indicated immunotherapies alone or in combination on thegrowth of 4T1 breast tumors in mice (n=6-8). Each line of FIG. 26Arepresents an individual mouse. FIG. 26B shows the tumor weight forindividual mice in each treatment. MSC-IL12+MSC-CCL21a shows bestefficacy compared to mice injected with naïve MSC. Treatment efficacywas also observed in the group treated with MSC-IFNb+MSC-CCL21a.

FIGS. 27A-27B are graphs that show immune profiles of each group treatedwith indicated engineered MSC. A consistent decrease in macrophagepopulation was observed after treating with MSC-IL12+MSC-CCL21a (FIG.27A). A general trend of increased infiltration in CD3+ population anddecreased infiltration in CD11b+ population was also observed whencompared to group treated with MSC-IL12+MSC-CCL21a against naïve MSC(FIG. 27A and FIG. 27B).

FIG. 28A-28B show the correlation of immune infiltration with tumorweight. Samples with low macrophage and dendritic cells have lower tumorburden (FIG. 28B, top center and top right). FIG. 28C shows the averagetumor weight from each group. Statistical significance was observed withboth MSC-IL12+MSC-CCL21a, or MSC-IFNb+MSC-CCL21a compared with naïveMSC.

FIG. 29 shows graphs combining the in vivo data from the colorectalcancer models above (FIG. 22A and FIG. 26A). The combined CT26 data fromFIG. 22A and FIG. 26A capture three groups: tumor only (PBS), treatedwith naïve MSC, and treated with MSC-IL12+MSC-CCL21a.

FIGS. 30A-30C also show combined data from FIG. 22A and FIG. 26A. Thegraphs show the average number of immune infiltration from the flowcytometry experiment data. Statistical significance was observed inCD8+T from FIG. 30A, demonstrating the ability of MSC-IL12+MSC-CCL21a torepolarize tumor microenvironment and allow more cytotoxic T cellinfiltration. Furthermore, there was a reduction in CD11b+ myeloidpopulation infiltration in the groups that were treated byMSC-IL12+MSC-CCL21a (FIG. 30B). The data collected using dendritic cellsand the macrophage population was statistical significance.

IL12 and CCL21a Therapy in Intraperitoneal and Subcutaneous Mouse Modelsof Colorectal Cancer

FIGS. 25A-25B include data from MSC-IL-12+CCL21a therapy inintraperitoneal and subcutaneous colorectal cancer mouse models. Threedifferent lots of a lentiviral transduced line was tested for MSC-IL12and CCL21a (TLOO8-3/4, TL019-01/02, and TL022-01/02; each TL numberrepresents one lot). FIG. 25A shows that all three lots ofMSC-IL12+MSC-CCL21a can reduce tumor burden in both subcutaneous andintraperitoneal model (first 5 graphs are from the SC model and last 3are from the IP model). Tumors from all mice were collected on day 11.FIG. 25B shows the average tumor weight from each group.

Example 6: MSC Combination Cytokine Therapy Methods

The following methods were used in experiments, as indicated.

Methods:

MSC Culturing

Bone-marrow derived C57BL/6 and Balb/C murine MSCs (mMSCs) werepurchased from Cyagen (Cat. No. MUBMX-01001 and MUCMX-01001,respectively). mMSC culturing media was composed of: MEM Corning Cat#10-022-CV (500 ml)+MSC FBS Gibco Cat #12662-029 (final conc 10%)+L-Glut(200 mM) Stem cell 07100 (Final conc 2 mM)+PenStrep 100×VWR Cat#97063-708 (Final conc 1×)+murine FGF Peprotech Cat #450-33-100uG (Finalconc-1:10,000 dilution). TrypLE Express was purchased(ThermoFisher—#12604021). PBS did not contain magnesium, calcium, orphenol red. mMSCs were passaged according to the protocol below:

-   -   1. mMSCs should be passaged at 70-90% confluency.    -   2. Aspirate media from dish/flask.    -   3. Rinse plate with PBS (e.g. 2 mL for 10 cm dish, 3 ml for 15        cm dish).    -   4. Add TrypLE Express (e.g. 2 mL for 10 cm dish, 3 ml for 15 cm        dish)    -   5. Incubate for 3-4 minutes at 37 degrees.    -   6. Knock plate on side to dislodge cells. Confirm by microscopy        that most cells have been dislodged.    -   7. Wash cells off plate using media (e.g. 8 mL for 10 cm dish).    -   8. Place cells in 15 conical and centrifuge 400×g for 5 min.    -   9. Aspirate media.    -   10. Resuspend cells in appropriate media and plate cells into        fresh plates/flasks.    -   Note: 70% confluent cells can be    -   split 1:3. 90% confluent cells can be split 1:4. Alternatively,        cells can be plated at 3000-5000 cells/cm2.

Bone-marrow derived human MSCs were purchased (RoosterBank-hBM-1M-XF,RoosterBio). Various hMSC culturing media were purchased: Xeno-free hMSCmedia—(RoosterBio—#KT-016); +FBS (serum-containing) hMSC media(Lonza—MSCGM media—#PT-3001). TrypLE Express was purchased(ThermoFisher—#12604021). PBS did not contain magnesium, calcium, orphenol red.

hMSCs were passaged according to the exemplary protocol below:

-   -   1. hMSCs should be passaged at 70-90% confluency.    -   2. Aspirate media from dish/flask.    -   3. Rinse plate with PBS (e.g. 2 mL for 10 cm dish).    -   4. Add TrypLE Express (e.g. 2 mL for 10 cm dish)    -   5. Incubate for 3-4 minutes at 37 degrees or 5 minutes RT.    -   6. Knock plate on side to dislodge cells. Confirm by microscopy        that most cells have been dislodged.    -   7. Wash cells off plate using Lonza MSCGM media (e.g. 8 mL for        10 cm dish).    -   8. Place cells in 15 conical and centrifuge 400×g for 5 min.    -   9. Aspirate media.    -   10. Resuspend cells in Rooster xeno-free media and plate cells        into fresh plates/flasks. Note: 70% confluent cells can be split        1:3. 90% confluent cells can be split 1:4. Alternatively, cells        can be plated at 3000-5000 cells/cm2.    -   hMSCs were thawed according to the exemplary protocol below:    -   1. Pre-warm hMSC media to 37°.    -   2. Remove hMSC aliquot from liquid nitrogen.    -   3. Thaw by holding the tube ½ submerged in 37° bath for 60-90        seconds, until ⅔ of the frozen sample has thawed.    -   4. Wipe the tube with 70% ethanol to sterilize tube.    -   5. Add 0.5 mL media to the cryotube, gently pipette 2-3 times,        and then transfer cells into 9 mL media (10 mL total) in 15 mL        conical tube.    -   6. Centrifuge 400×g for 5 min.    -   7. Aspirate media, and then gently resuspend pellet in        appropriate volume of Rooster xeno-free media. Plate cells at a        concentration of 3000-5000 cells/cm2.

Lentiviral Production

Lentivirus was produced using: Lenti-X 293T packaging cell line(Clontech, Cat #632180); LX293T Complete growth medium, withoutantibiotics; DMEM, hi-glucose; 1 mM Sodium Pyruvate; 10% FBS,heat-inactivated; Opti-Mem I Reduced Serum Media (Gibco/Thermo Fisher;Cat #31985); FuGene HD (Promega, Cat #E2311); Envelope, Packaging, andTransfer Vector plasmids; VSV-G-pseudotyped envelope vector (pMD2.G);Packaging vector that contains Gag, Pol, Rev, and Tat that can be usedwith 2nd and 3rd generation transfer vectors (psMAX2). 293T(FT) cellsfrom 90% confluent 10 cm dishes were lifted and dispensed at 1:3dilution late in the afternoon the day before transfection and incubatedcells as normal overnight at 37° C., 5% CO2 (cells should be 60-85%confluent the next day at time of transfection).

A transfection reaction was prepped for each 10 cm dish according to theprotocol below:

-   -   1. Prep transfection reaction for each 10 cm dish in a separate        1.7 mL tube.    -   2. Add 900 uL Opti-Mem I at RT.    -   3. Add 9 ug vector backbone (containing gene of interest) per        reaction.    -   4. Add Bug packaging vector per reaction.    -   5. Add 1 ug envelope vector per reaction (pMD2.G).    -   6. Mix thoroughly by quickly vortexing for 3 seconds.    -   7. Add 55 uL Fugene HD per reaction.    -   8. Mix by quickly pipetting up and down 20-30 times.    -   9. Let sit at RT for 10 min (allowing DNA complexes to form).    -   10. Slowly add mixture in dropwise manner around the dish, then        mix by gently rocking back-forth and up-down for 5-10 seconds        (do not swirl).    -   11. Place dish into virus incubator.

Viral supernatants were harvested on days 2 and 3 using a serologicalpipette. Cellular debris was removed using a Millipore steriflip 0.45 umfilters. A Lenti-X Concentrator (Cat. Nos. 631231 & 631232) was usedaccording to the protocol: 1) Combine 1 volume of Lenti-X Concentratorwith 3 volumes of clarified supernatant. Mix by gentle inversion; 2)Incubate mixture on ice or at 4° C. for 30 minutes to overnight; (3)Centrifuge sample at 1,500×g for 45 minutes at 4° C.; (4) Carefullyremove and discard supernatant, taking care not to disturb the pellet;(5) Gently resuspend the pellet in 1/10 to 1/100th of the originalvolume using sterile PBS+0.1% BSA.

Vectors

Cytokine expression cassettes were cloned into a pL17D, the vector mapof which is shown in FIG. 31 with salient features annotated; e.g., aSFFV promoter; a FLAG and MYC epitope tag; LTRs, etc.

Lentiviral Transduction

Murine MSCs were seeded in 6-well plates and infected when cells were50% confluent. Virus was added at the appropriate MOI and incubated for3 hours to transduce cells. Following infection, fresh media was addedto the cells.

Human MSCs were transduced following the exemplary protocol below:

-   -   1. 200,000 human MSCs were plated in each well of 6-well plate,        in 2 mL xeno-free human MSC media.    -   2. After 2 hours, the media was removed and replaced with 1 mL        of PBS.    -   3. Appropriate amount of virus was added to each well, as        indicate by MOI below, and cells were incubated with virus for 3        hours with occasional rocking, at 37 degrees and 5% CO2.    -   4. Virus was removed after 3 hours, plates were washed with        media, and then the MSCs were cultured normally (as noted above)        until cells reached senescence. Cells were counted at each        passage, so that total cell numbers could be determined.

Example 7: MSC Combination Cytokine Therapy (CT26)

In the following example, balb/c mMSCs were engineered to expressvarious cytokines using the lentiviral transduction method described inExample 6.

CT26 tumor cells (5×10⁴ cells in 100 μl) modified to constitutivelyexpress luciferase enzyme (Cat no: CL043, Lot no: CL-IM147 Imanis LifeSciences) were injected into the peritoneal space of immunocompetentbalb/c (age 6-8 weeks). One week after tumor implantation, tumor burdenwas measured by luciferase imaging (BLI) using an AMI imager. Mice wererandomized into treatment groups and treated with intraperitoneallydelivered mMSCs (1×10⁶) expressing effector molecules as single agent oras a combination of mMSCs to deliver a combination of agents.MSC-Flag-Myc and PBS were used as a negative control. Tumor burden wasassessed at day 12 and 17. Bioluminescent signal (photons/second) wasnormalized for each individual mouse relative to the initial signal(pre-treatment). Reduction of BLI signal by more than 100 fold (0.01)was equivalent to a complete cure (no tumor was evident at the time ofnecropsy). As shown in FIG. 32, MSCs engineered to express differenteffector molecules either alone or in combination demonstrated efficacyin reducing CT26 tumor burden in an IP tumor model as assessed by BLIlevels.

Example 8: MSC Combination Cytokine Therapy (B16F10)

In the following example, C57BL/6 mMSCs were engineered to expressvarious cytokines using the lentiviral transduction method described inExample 6.

B16F10 tumor cells (5×10⁴ cells in 100 μl) modified to constitutivelyexpress luciferase enzyme (B16F10-Fluc-Puro Cat #:CL052, lot #: CL-IM150Imanis Life Sciences) were injected into the peritoneal space ofimmunocompetent C57BL/6 (age 6-8 weeks). One week after tumorimplantation, tumor burden was measured by luciferase imaging (BLI)using an AMI imager. Mice were randomized into treatment groups andtreated with intraperitoneally delivered mMSCs (1×10⁶) expressingeffector molecules as single agent or as a combination of mMSCs todeliver a combination of agents. MSC-Flag-Myc and PBS were used as anegative control. Tumor burden was assessed at day 12 and 17.Bioluminescent signal (photons/second) was normalized for eachindividual mouse relative to the initial signal (pre-treatment).Reduction of BLI signal by more than 100 fold (0.01) was equivalent to acomplete cure (no tumor was evident at the time of necropsy). As shownin FIG. 33, MSCs engineered to express different effector moleculeseither alone or in combination demonstrated efficacy in reducing B16F10tumor burden in an IP tumor model as assessed by BLI levels.

Example 9: Engineered Human MSC Cytokine Production

In the following example, bone-marrow derived hMSCs (derived from 3human volunteer healthy donors) were engineered to express human IL12(p70) and human CCL21a from a single lentiviral expression vector usingthe lentiviral transduction method described in Example 6. Thelentiviral expression vector (schematic vector map of which is shown inFIG. 34) used a 2A ribosome skipping elements to express both cytokinesfrom a single transcript.

As shown in FIG. 35, engineered hMSCs were able to produce both hIL12(FIG. 35A) and hCCL21a (FIG. 35B), as assessed by cytokine ELISA.Notably, protein secretion was correlated with the amount of viralparticles (MOI) used during the transduction of MSCs.

Example 10: Engineered Human MSC Functional Assessment

In the following example, bone-marrow derived hMSCs were engineered toexpress human IL12 (p70) using the lentiviral transduction methoddescribed in Example 6. Engineered hMSCs were co-cultured into 0.4 μmtranswell inserts with human T-cells isolated from healthy blood donors(a schematic representation of the transwell assay is shown in FIG.36A). To assess IL12 induced Th1 polarization on activated naïveT-cells, IFNγ production by T-cells was measured by ELISA on thesupernatant collected from the lower compartment (T-cells). As shown inFIG. 36B, IFNγ production was increased in a MOI dose-dependent mannerby co-culturing CD3 T-cells with hMSCs expressing IL12p70.

Example 11: MSCs Home to Tumors in an IP Model

In the following example, balb/c MSCs (2×10⁶ cells) expressing fLUC wereinjected IP into CT-26 IP tumor-bearing mice. Mice were euthanized andtissues were collected 24 hours after injection. As shown in FIG. 37,fLUC-MSCs were significantly enriched in the tumors as detected bybioluminescence imaging (images shown in FIG. 37A, quantification ofimages in FIG. 37B), quantitative real time PCR (FIG. 37C), andfluorescence microscopy against firefly luciferase (FIG. 37D).

Additionally, C57Bl/6 mice were implanted with 5×10⁴ B16F10-fLUC cellsIP. 7 days after tumor implantation, 1×10⁶ C57Bl/6 murine BM-MSCsengineered to express Nanoluc-EGFP were injected IP. Mice wereeuthanized at 24 hours post injection of MSCs and peritoneal organs(stomach, kidney, liver, colon, spleen, pancreas, omentum/tumor, ovariesand Fallopian tubes) were imaged ex-vivo for nanoluc signaling (NanoGloSubstrate Kit, Vendor: Promega, Catalog No.: N1110). As shown in FIG.37E, murine MSC nanoluc signal was preferentially enriched in the tumorcompared to the other organs in the peritoneal cavity in a B16F10 tumormodel.

Example 12: IL12 Producing MSCs Reduce CT26 Tumor Burden in an IP Model

In the following example, balb/c mMSCs were engineered to express murineIL12p70 using the lentiviral transduction method described in Example 6.

CT26 tumor cells (5×10⁴ cells in 100 μl) modified to constitutivelyexpress luciferase enzyme (Cat no: CL043, Lot no: CL-IM147 Imanis LifeSciences) were injected into the peritoneal space of immunocompetentbalb/c (age 6-8 weeks). One week after tumor implantation, tumor burdenwas measured by luciferase imaging (BLI) using an AMI imager. Mice wererandomized into treatment groups and treated with intraperitoneallydelivered mMSCs (1×10⁶ cells) expressing IL12p70. MSC-Flag-Myc and PBSwere used as a negative control. As shown in FIG. 38, IL12p70 expressingMSCs led to reduction in tumor burden as assessed by BLI (top panels andbottom left panel) and a complete elimination of detectableintraperitoneal tumors by tumor weight (bottom right panel) in a CT26model.

Example 13: IL12 Producing MSCs Reduce B16F10 Tumor Burden in an IPModel

In the following example, C57BL/6 mMSCs were engineered to expressmurine IL12p70 using the lentiviral transduction method described inExample 6.

B16F10 tumor cells (5×10⁴ cells in 100 μl) modified to constitutivelyexpress luciferase enzyme (B16F10-Fluc-Puro Cat #:CL052, lot #: CL-IM150Imanis Life Sciences) were injected into the peritoneal space ofimmunocompetent C57BL/6 (age 6-8 weeks). One week after tumorimplantation, tumor burden was measured by luciferase imaging (BLI)using an AMI imager. Mice were randomized into treatment groups andtreated with intraperitoneally delivered mMSCs 1×10⁶ expressing IL12p70.MSC-Flag-Myc and PBS were used as a negative control. As shown in FIG.39, IL12p70 expressing MSCs led to reduction in tumor burden as assessedby BLI (top panels and bottom left panel) and a complete elimination ofdetectable intraperitoneal tumors by tumor weight (bottom right panel)in a B16F10 model.

Example 14: MSCs Producing IL12 and CCL21a Reduce Tumor Burden andProlong Survival in a CT26 IP Tumor Model

In the following example, balb/c mMSCs were engineered to express murineIL12 (p70) and murine CCL21a from a single lentiviral expression vector.The lentiviral expression vector used a 2A ribosome skipping elements toexpress both cytokines from a single transcript using the lentiviraltransduction method described in Example 6.

CT26 tumor cells (1×10⁶ cells) modified to constitutively expressluciferase enzyme (Cat no: CL043, Lot no: CL-IM147 Imanis Life Sciences)were injected into the peritoneal space of immunocompetent balb/c mice(age 6-8 weeks). One week after tumor implantation, tumor burden wasmeasured by luciferase imaging (BLI) using an AMI imager. Mice wererandomized into treatment groups and treated with intraperitoneallydelivered mMSCs 1×10⁶ expressing IL12p70 and CCL21a by the same MSC(“MSC-IL-12p70_2A_CCL21a”). MSC-Flag-Myc and PBS were used as a negativecontrol. As shown in FIG. 40, IL12p70/CCL21a expressing MSCs led toreduction in tumor burden as assessed by BLI (top panels and bottom leftpanel) and a complete elimination of detectable intraperitoneal tumorsby tumor weight (bottom right panel) in a CT26 model. FIG. 40Ademonstrates the mean tumor burden as assessed by BLI for PBS treated(circle), MSC-Flag-Myc (“Naïve MSC” square), and IL12p70/CCL21aexpressing MSCs (triangle). FIG. 40B demonstrates the tumor burden inindividual mice as assessed by BLI for PBS treated, MSC-Flag-Myc (“NaïveMSC”), and IL12p70/CCL21a expressing MSCs (left, middle, and rightpanels, respectively). Notably, as shown in FIG. 40C, treatment withIL12p70/CCL21a expressing MSCs led to prolonged survival (100% survivalgreater than 90 days), while control treated mice all died or wereeuthanized by Day 20.

Example 15: MSCs Producing IL12 and IL21 Reduce Tumor Burden and ProlongSurvival in a B16F10 IP Tumor Model

In the following example, C57BL/6 mMSCs were engineered to expressmurine IL12 (p70) or murine IL21 (i.e., each MSC engineered to expressonly a single agent) using the lentiviral transduction method describedin Example 6.

B16F10 tumor cells (5×10⁴ cells in 100 μl) modified to constitutivelyexpress luciferase enzyme (B16F10-Fluc-Puro Cat #:CL052, lot #: CL-IM150Imanis Life Sciences) were injected into the peritoneal space ofimmunocompetent C57BL/6 (age 6-8 weeks). One week after tumorimplantation, tumor burden was measured by luciferase imaging (BLI)using an AMI imager. Mice were randomized into treatment groups andtreated with intraperitoneally delivered mMSCs (1×10⁶ cells) expressingIL12p70 in combination with mMSCs (1×10⁶ cells) expressing IL21, ormMSCs (1×10⁶ cells) expressing IL12p70 alone. MSC-Flag-Myc and PBS wereused as a negative control. As shown in FIG. 41, treatment with IL12p70expressing MSCs led to prolonged survival relative to control treatedmice but all mice still all died or were euthanized by Day 50. Incontrast, treatment with IL12p70 expressing MSCs in combination withIL21 expressing MSCs led to prolonged survival relative to treatmentwith IL12p70 expressing MSCs (60% survival past 60 days). Thus, IL21expression by MSCs enhanced the efficacy of IL12p70 expressing MSCs.

Example 16: Allogeneic MSCs Producing IL12 and CCL21a Reduce TumorBurden and Prolong Survival in a CT26 IP Tumor Model

In the following example, balb/c mMSCs (syngeneic) and C57BL/6 mMSCs(allogeneic) were engineered to express murine IL12 (p70) and murineCCL21a from a single lentiviral expression vector. The lentiviralexpression vector used a 2A ribosome skipping elements to express bothcytokines from a single transcript using the lentiviral transductionmethod described in Example 6.

CT26 tumor cells (1×10⁶ cells) modified to constitutively expressluciferase enzyme (Cat no: CL043, Lot no: CL-IM147 Imanis Life Sciences)were injected into the peritoneal space of immunocompetent balb/c mice(age 6-8 weeks). One week after tumor implantation, tumor burden wasmeasured by luciferase imaging (BLI) using an AMI imager. Mice wererandomized into treatment groups and treated with intraperitoneallydelivered mMSCs (1×10⁶ cells) expressing IL12p70 and CCL21a by the sameMSC (“MSC-IL12+CCL21”). Both balb/c control mMSCs (syngeneic) andC57BL/6 control mMSCs (allogeneic) were engineered to expressMSC-Flag-Myc (“Naïve”). PBS was also used as a negative control. Asshown in FIG. 1, both syngeneic and allogeneic MSCs expressingIL12p70/CCL21a led to reduction in tumor burden as assessed by BLI in aCT26 model, while control treatments did not. Additionally, mice thatwere previously treated with mMSCs expressing IL12p70 and CCL21a in bothsyngeneic and allogeneic models and were determined to be tumor free for90 days were subsequently challenged with CT26 tumor cells (0.5×10⁶cells in 100 μl PBS) implanted subcutaneously in the thigh, asschematized in FIG. 2A. As shown in FIG. 2B, tumor free mice rejectedthe tumor implant in contrast to naïve control mice where the tumorbecame established. Thus, treatment with MSCs expressing IL12p70/CCL21aled to prolonged tumor burden reduction as well as immunological memory.

Example 17: MSCs Producing IL12 and CCL21a Demonstrate Enhanced GrowthRelative to Unmodified Cells

In the following example, human MSCs from 3 different donors wereengineered at different multiplicity of infections (MOIs) to express andsecrete human IL-12 and human CCL21a from a single lentiviral expressionvector. The lentiviral expression vector used a 2A ribosome skippingelements to express both cytokines from a single transcript using thelentiviral transduction method described in Example 6.

As shown in FIG. 42, the genetically engineered MSCs (MOI=95000, 9500,or 950) exhibited enhanced cell expansion and growth compared to thenon-genetically engineered human MSCs (MOI=0) in the three donors tested(FIG. 42A, Donor 1; FIG. 42B, Donor 2; FIG. 42C, Donor 3). Human MSCsgenetically engineered with lentivirus to express GFP did not show asimilar enhanced cell expansion or growth phenotype (data not shown).

Example 18: Selection of Promoter for Sustained Protein Expression inHuman Bone-Marrow MSCs (BM-MSCs)

In the following example, various promoters were tested for drivingexpression of a reporter EGFP construct in human MSCs. Promoters testedwere CMV, SFFV, EF1a, EF1a-LTR, EFS, MND, PGK, UbC (see Table 4). Cellswere transduced using equivalent MOI (multiplicity of infection) usingthe lentiviral transduction method described in Example. EGFP percentageand Median Fluorescence Intensity (MFI) were quantified over serialpassages using flow cytometry.

As shown in FIG. 43, two independent human BM-MSC cell lines from 2different donors (top and bottom row, respectively were engineered andpercent GFP (left panels) and MFI (right panels) of engineered cells wasassessed at day 25 post transduction. The SFFV promoter demonstrated GFPexpression in both cell lines by both GFP percentage and MFI.

As shown in FIG. 44, EGFP MFI was tracked over time (day 7 to day 28post-transduction) for either the two independent human BM-MSC celllines individually (left panel) or with data from the two independenthuman BM-MSC cell lines combined (right panel). Protein expression wasstable over time during more than 28 days. Additionally, in comparisonto EF1a promoters, SFFV promoter consistently drove almost ten-fold moreprotein expression as quantified by MFI.

Example 19: Engineering Human MSCs to Produce IL12 and IL21

In the following example, human bone-marrow MSCs were stably transducedto express IL12p70 and IL21 from various constructs using the lentiviraltransduction method described in Example 6. Cells were expanded for 3 to4 passages post-transduction and 0.2×10⁶ cells were seeded in 6-wellplates in 4 mL of media. Conditioned media was collected after 24 hoursand ELISAs were performed to determine the IL-12 and IL-21concentrations produced.

Various constructs were tested with different combinations and/orarrangements of promoter-signal sequence 1-cytokine 1-2A linker-signalsequence 2-cytokine 2. The combinations tested are described below inTable 7. Specific details of construct SB00880 are presented below inTable 8.

TABLE 7 IL-12 and IL-21 Expression Constructs Codon Construct Opti- NameBack- mi- (SB#) Promoter Insert bone zation SB00743 SFFV_1 IL12ss-IL12pL23d None SB00763 EFa1 IL12ss-IL12-fT2A*-IL21ss-IL21 pL40g None (pEF6)SB00765 EFa1 IL12ss-IL12-fT2A-IL12ss-IL21 pL40g None (pEF6) SB00766 EFa1IL12ss-IL12-fT2A-IL8ss-IL21 pL40g None (pEF6) SB00767 EF1aIL12ss-IL12-fT2A-IL21 pL40g None (pEF6) SB00768 EFa1IL21ss-IL21-fT2A-IL12ss-IL12 pL40g None (pEF6) SB00769 EFa1IL12ss-IL21-fT2A-IL12ss-IL12 pL40g None (pEF6) SB00770 EFa1IL6ss-IL21-fT2A-IL12ss-IL12 pL40g None (pEF6) SB00771 EF1aIL8ss-IL21-fT2A-IL12ss-IL12 pL40g None (pEF6) SB00772 EF1aIL21ss-IL21-fT2A-IL12 pL40g None (pEF6) SB00773 EF1aIL12ss-IL21-fT2A-IL12 pL40g None (pEF6) SB00774 EF1aIL6ss-IL21-fT2A-IL12 pL40g None (pEF6) SB00775 EF1a IL8ss-IL21-fT2A-IL12pL40g None (pEF6) SB00772 EF1a IL21ss-IL21-fT2A-IL12 pL40g None (pEF6)SB00620 SFFV_1 IL2ss-IL21 pL17d None SB00838 SFFV_1IL12ss-IL12-fT2A-IL21ss-IL21 pL41g None SB00839 SFFV_1IL12ss-IL12-fT2A-IL8ss-IL21 pL41g None SB00840 SFFV_1IL12ss-IL12-fT2A-IL21 pL41g None SB00841 SFFV_1IL21ss-IL21-fT2A-IL12ss-IL12 pL41g None SB00843 SFFV_1IL21ss-IL21-fT2A-IL12 pL41g None SB00844 SFFV_1 IL8ss-IL21-fT2A-IL12pL41g None SB00868 SFFV_1 IL12ss-IL12 pL41g Yes SB00870 EF1aIL12ss-IL12-fT2A-IL21ss-IL21 pL40g Yes (pEF6) SB00872 EF1aIL12ss-IL12-fT2A-IL21 pL40g Yes (pEF6) SB00869 EF1aIL21ss-IL21-fT2A-IL12ss-IL12 pL40g Yes (pEF6) SB00871 EF1aIL21ss-IL21-fT2A-IL12 pL40g Yes (pEF6) SB00879 SFFV_1IL21ss-IL21-fT2A-IL12ss-IL12 pL41g Yes SB00880 SFFV_1IL12ss-IL12-fT2A-IL21ss-IL21 pL41g Yes SB00881 SFFV_1IL21ss-IL21-fT2A-IL12 pL41g Yes SB00882 SFFV_1 IL12ss-IL12-fT2A-IL21pL41g Yes SB00862 SFFV_1 IL21ss-IL21 pL23d None SB00863 SFFV_1IL2ss-IL21 pL41g Yes SB00968 SFFV_1 IL2ss-IL21-fT2A-IL12ss-IL12 pL41gYes SB00969 SFFV_1 IL8ss-IL21-fT2A-IL12ss-IL12 pL41g Yes SB00970 SFFV_1IL12ss-IL12-fT2A-IL2ss-IL21 pL41g Yes SB00971 SFFV_1IL12ss-IL12-fT2A-IL8ss-IL21 pL41g Yes SB00862 + SFFV_1 Co-transductionIL12 + IL21 pL41g None SB00743 SB00868 + SFFV_1 Co-transduction IL12 +IL21 pL41g Yes 863 (IL2ssIL21) *fT2A refers to Furin-T2A

TABLE 8 SB00880 Expression Construct SequencesSFFV promoter (SEQ ID NO: 17)GTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGCCCGGGHuman IL-12 signal sequence; codon optimized(nucleic acid) (SEQ ID NO: 32)ATGTGCCATCAGCAACTCGTCATCTCCTGGTTCTCCCTTGTGTTCCT CGCTTCCCCTCTGGTCGCCHuman IL-12 signal sequence (amino acid) (SEQ ID NO: 112)MCHQQLVISWFSLVFLASPLVA Human IL-12 protein without signal sequence;codon optimized (nucleic acid) (SEQ ID NO: 136)ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAGCTGGATTGGTACCCGGACGCCCCTGGAGAAATGGTCGTGCTGACTTGCGATACGCCAGAAGAGGACGGCATAACCTGGACCCTGGATCAGAGCTCCGAGGTGCTCGGAAGCGGAAAGACCCTGACCATTCAAGTCAAGGAGTTCGGCGACGCGGGCCAGTACACTTGCCACAAGGGTGGCGAAGTGCTGTCCCACTCCCTGCTGCTGCTGCACAAGAAAGAGGATGGAATCTGGTCCACTGACATCCTCAAGGACCAAAAAGAACCGAAGAACAAGACCTTCCTCCGCTGCGAAGCCAAGAACTACAGCGGTCGGTTCACCTGTTGGTGGCTGACGACAATCTCCACCGACCTGACTTTCTCCGTGAAGTCGTCACGGGGATCAAGCGATCCTCAGGGCGTGACCTGTGGAGCCGCCACTCTGTCCGCCGAGAGAGTCAGGGGAGACAACAAGGAATATGAGTACTCCGTGGAATGCCAGGAGGACAGCGCCTGCCCTGCCGCGGAAGAGTCCCTGCCTATCGAGGTCATGGTCGATGCCGTGCATAAGCTGAAATACGAGAACTACACTTCCTCCTTCTTTATCCGCGACATCATCAAGCCTGACCCCCCCAAGAACTTGCAGCTGAAGCCACTCAAGAACTCCCGCCAAGTGGAAGTGTCTTGGGAATATCCAGACACTTGGAGCACCCCGCACTCATACTTCTCGCTCACTTTCTGTGTGCAAGTGCAGGGAAAGTCCAAACGGGAGAAGAAAGACCGGGTGTTCACCGACAAAACCTCCGCCACTGTGATTTGTCGGAAGAACGCGTCAATCAGCGTCCGGGCGCAGGATAGATACTACTCGTCCTCCTGGAGCGAATGGGCCAGCGTGCCTTGTTCCGGTGGCGGATCAGGCGGAGGTTCAGGAGGAGGCTCCGGAGGAGGTTCCCGGAACCTCCCTGTGGCAACCCCCGACCCTGGAATGTTCCCGTGCCTACACCACTCCCAAAACCTCCTGAGGGCTGTGTCGAACATGTTGCAGAAGGCCCGCCAGACCCTTGAGTTCTACCCCTGCACCTCGGAAGAAATTGATCACGAGGACATCACCAAGGACAAGACCTCGACCGTGGAAGCCTGCCTGCCGCTGGAACTGACCAAGAACGAATCGTGTCTGAACTCCCGCGAGACAAGCTTTATCACTAACGGCAGCTGCCTGGCGTCGAGAAAGACCTCATTCATGATGGCGCTCTGTCTTTCCTCGATCTACGAAGATCTGAAGATGTATCAGGTCGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCGAAGCGGCAGATCTTCCTGGACCAGAATATGCTCGCCGTGATTGATGAACTGATGCAGGCCCTGAATTTCAACTCCGAGACTGTGCCTCAAAAGTCCAGCCTGGAAGAACCGGACTTCTACAAGACCAAGATCAAGCTGTGCATCCTGTTGCACGCTTTCCGCATTCGAGCCGTGACCATTGACCGCGTGATGTCCTACCTGAAC GCCAGTHuman IL-12 protein without signal sequence(amino acid) (SEQ ID NO: 137); p35 subunitin bold; p40 subunit in italicsIWELKKDVYVVELDWYPDAPGLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVETDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGSGGGSGGGSGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNENSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASHuman IL-12 protein with signal sequence (aminoacid) (SEQ ID NO: 138); p35 subunit in bold; p40 subunit in italicsMCHQQLVISWFSLVFLASPLVAIWELKLDVYVVELDWYPDAPGLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCE4KNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVETDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGSGGGSGGGSGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILL HAFRIRAVTIDRVMSYLNASFurin-T2A Linker (nucleic acid) (SEQ ID NO: 139)AGACGGAAACGCGGAAGCGGAGAGGGCAGAGGCTCGCTGCTTACATGCGGGGACGTGGAAGAGAACCCCGGTCCGFurin-T2A Linker (amino acid) (SEQ ID NO: 140) RRKRGSGEGRGSLLTCGDVEENPGPHuman IL-21 signal sequence; codon optimized(nucleic acid) (SEQ ID NO: 55)ATGGAACGCATTGTGATCTGCCTGATGGTCATCTTCCTGGGCACCTT AGTGCACAAGTCGAGCAGCHuman IL-21 signal sequence (amino acid) (SEQ ID NO: 135)MERIVICLMVIFLGTLVHKSSS Human IL-21 protein without signal sequence;codon optimized (nucleic acid) (SEQ ID NO: 141)CAGGGACAGGACAGGCACATGATTAGAATGCGCCAGCTCATCGATATCGTGGACCAGTTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCGGCCCCCGAAGATGTGGAAACCAATTGCGAATGGTCGGCATTTTCCTGCTTTCAAAAGGCACAGCTCAAGTCCGCTAACACCGGGAACAACGAACGGATCATCAACGTGTCCATCAAAAAGCTGAAGCGGAAGCCTCCCTCCACCAACGCCGGACGGAGGCAGAAGCATAGGCTGACTTGCCCGTCATGCGACTCCTACGAGAAGAAGCCGCCGAAGGAGTTCCTGGAGCGGITCAAGTCGCTCCTGCAAAAGATGATTCATCAGCACCTGTCCTCCCGGACTCAT GGGTCTGAGGATTCAHuman IL-21 protein without signal sequence(amino acid) (SEQ ID NO: 142)QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSHuman IL-21 protein with signal sequence (amino acid) (SEQ ID NO: 143)MERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQH LSSRTHGSEDSSB00880 Cassette (SFFV-IL12ss-IL12-fT2A- IL21ss-IL21) (SEQ ID NO: 144)GTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTITCGGCCCCGGCCCGGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGCCCGGGGGATCCGCGGAATTCGCCGCCACCATGTGCCATCAGCAACTCGTCATCTCCTGGTTCTCCCTTGTGTTCCTCGCTTCCCCTCTGGTCGCCATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAGCTGGATTGGTACCCGGACGCCCCTGGAGAAATGGTCGTGCTGACTTGCGATACGCCAGAAGAGGACGGCATAACCTGGACCCTGGATCAGAGCTCCGAGGTGCTCGGAAGCGGAAAGACCCTGACCATTCAAGTCAAGGAGTTCGGCGACGCGGGCCAGTACACTTGCCACAAGGGTGGCGAAGTGCTGTCCCACTCCCTGCTGCTGCTGCACAAGAAAGAGGATGGAATCTGGTCCACTGACATCCTCAAGGACCAAAAAGAACCGAAGAACAAGACCTTCCTCCGCTGCGAAGCCAAGAACTACAGCGGTCGGTTCACCTGTTGGTGGCTGACGACAATCTCCACCGACCTGACTTTCTCCGTGAAGTCGTCACGGGGATCAAGCGATCCTCAGGGCGTGACCTGTGGAGCCGCCACTCTGTCCGCCGAGAGAGTCAGGGGAGACAACAAGGAATATGAGTACTCCGTGGAATGCCAGGAGGACAGCGCCTGCCCTGCCGCGGAAGAGTCCCTGCCTATCGAGGTCATGGTCGATGCCGTGCATAAGCTGAAATACGAGAACTACACTTCCTCCTTCTTTATCCGCGACATCATCAAGCCTGACCCCCCCAAGAACTTGCAGCTGAAGCCACTCAAGAACTCCCGCCAAGTGGAAGTGTCTTGGGAATATCCAGACACTTGGAGCACCCCGCACTCATACTTCTCGCTCACTTTCTGTGTGCAAGTGCAGGGAAAGTCCAAACGGGAGAAGAAAGACCGGGTGTTCACCGACAAAACCTCCGCCACTGTGATTTGTCGGAAGAACGCGTCAATCAGCGTCCGGGCGCAGGATAGATACTACTCGTCCTCCTGGAGCGAATGGGCCAGCGTGCCTTGTTCCGGTGGCGGATCAGGCGGAGGTTCAGGAGGAGGCTCCGGAGGAGGTTCCCGGAACCTCCCTGTGGCAACCCCCGACCCTGGAATGTTCCCGTGCCTACACCACTCCCAAAACCTCCTGAGGGCTGTGTCGAACATGTTGCAGAAGGCCCGCCAGACCCTTGAGTTCTACCCCTGCACCTCGGAAGAAATTGATCACGAGGACATCACCAAGGACAAGACCTCGACCGTGGAAGCCTGCCTGCCGCTGGAACTGACCAAGAACGAATCGTGTCTGAACTCCCGCGAGACAAGCTTTATCACTAACGGCAGCTGCCTGGCGTCGAGAAAGACCTCATTCATGATGGCGCTCTGTCITTCCTCGATCTACGAAGATCTGAAGATGTATCAGGTCGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCGAAGCGGCAGATCTTCCTGGACCAGAATATGCTCGCCGTGATTGATGAACTGATGCAGGCCCTGAATTTCAACTCCGAGACTGTGCCTCAAAAGTCCAGCCTGGAAGAACCGGACTTCTACAAGACCAAGATCAAGCTGTGCATCCTGTTGCACGCTTTCCGCATTCGAGCCGTGACCATTGACCGCGTGATGTCCTACCTGAACGCCAGTAGACGGAAACGCGGAAGCGGAGAGGGCAGAGGCTCGCTGCTTACATGCGGGGACGTGGAAGAGAACCCCGGTCCGATGGAACGCATTGTGATCTGCCTGATGGTCATCTTCCTGGGCACCTTAGTGCACAAGTCGAGCAGCCAGGGACAGGACAGGCACATGATTAGAATGCGCCAGCTCATCGATATCGTGGACCAGTTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCGGCCCCCGAAGATGTGGAAACCAATTGCGAATGGTCGGCATTTTCCTGCTTTCAAAAGGCACAGCTCAAGTCCGCTAACACCGGGAACAACGAACGGATCATCAACGTGTCCATCAAAAAGCTGAAGCGGAAGCCTCCCTCCACCAACGCCGGACGGAGGCAGAAGCATAGGCTGACTTGCCCGTCATGCGACTCCTACGAGAAGAAGCCGCCGAAGGAGTTCCTGGAGCGGTTCAAGTCGCTCCTGCAAAAGATGATTCATCAGCACCTGTCCTCCCGGACTCATGGGTCTGAGGATTCATG ASB00880 Full Vector (PL41g + SB00880 Cassette) (SEQ ID NO: 145)TGACTCCTGCGCAGTCCAAAAAAAAAGGCTCCAAAAGGAGCCTTTAATTGTATCGGTGGGCCCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACGTGGTTTAAACAAATAGTCAAAAGCCTCCGGCGACTAGTCGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTIGTTITGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGGGTCTCTCTGGTTAGACCAGATTTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGCAGAGTACGCCAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAAAATAGCGGCCGCCACAATTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAATTTTCGGGGGATCCGTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGCCCGGGGGATCCGCGGAATTCGCCGCCACCATGTGCCATCAGCAACTCGTCATCTCCTGGTTCTCCCTTGTGTTCCTCGCTTCCCCTCTGGTCGCCATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAGCTGGATTGGTACCCGGACGCCCCTGGAGAAATGGTCGTGCTGACTTGCGATACGCCAGAAGAGGACGGCATAACCTGGACCCTGGATCAGAGCTCCGAGGTGCTCGGAAGCGGAAAGACCCTGACCATTCAAGTCAAGGAGTTCGGCGACGCGGGCCAGTACACTTGCCACAAGGGTGGCGAAGTGCTGTCCCACTCCCTGCTGCTGCTGCACAAGAAAGAGGATGGAATCTGGTCCACTGACATCCTCAAGGACCAAAAAGAACCGAAGAACAAGACCTTCCTCCGCTGCGAAGCCAAGAACTACAGCGGTCGGTTCACCTGTTGGTGGCTGACGACAATCTCCACCGACCTGACTTTCTCCGTGAAGTCGTCACGGGGATCAAGCGATCCTCAGGGCGTGACCTGTGGAGCCGCCACTCTGTCCGCCGAGAGAGTCAGGGGAGACAACAAGGAATATGAGTACTCCGTGGAATGCCAGGAGGACAGCGCCTGCCCTGCCGCGGAAGAGTCCCTGCCTATCGAGGTCATGGTCGATGCCGTGCATAAGCTGAAATACGAGAACTACACTTCCTCCTTCTTTATCCGCGACATCATCAAGCCTGACCCCCCCAAGAACTTGCAGCTGAAGCCACTCAAGAACTCCCGCCAAGTGGAAGTGTCTTGGGAATATCCAGACACTTGGAGCACCCCGCACTCATACTTCTCGCTCACTTTCTGTGTGCAAGTGCAGGGAAAGTCCAAACGGGAGAAGAAAGACCGGGTGTTCACCGACAAAACCTCCGCCACTGTGATTTGTCGGAAGAACGCGTCAATCAGCGTCCGGGCGCAGGATAGATACTACTCGTCCTCCTGGAGCGAATGGGCCAGCGTGCCTTGTTCCGGTGGCGGATCAGGCGGAGGTTCAGGAGGAGGCTCCGGAGGAGGTTCCCGGAACCTCCCTGTGGCAACCCCCGACCCTGGAATGTTCCCGTGCCTACACCACTCCCAAAACCTCCTGAGGGCTGTGTCGAACATGTTGCAGAAGGCCCGCCAGACCCTTGAGTTCTACCCCTGCACCTCGGAAGAAATTGATCACGAGGACATCACCAAGGACAAGACCTCGACCGTGGAAGCCTGCCTGCCGCTGGAACTGACCAAGAACGAATCGTGTCTGAACTCCCGCGAGACAAGCTTTATCACTAACGGCAGCTGCCTGGCGTCGAGAAAGACCTCATTCATGATGGCGCTCTGTCTTTCCTCGATCTACGAAGATCTGAAGATGTATCAGGTCGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCGAAGCGGCAGATCTTCCTGGACCAGAATATGCTCGCCGTGATTGATGAACTGATGCAGGCCCTGAATTTCAACTCCGAGACTGTGCCTCAAAAGTCCAGCCTGGAAGAACCGGACTTCTACAAGACCAAGATCAAGCTGTGCATCCTGTTGCACGCTTTCCGCATTCGAGCCGTGACCATTGACCGCGTGATGTCCTACCTGAACGCCAGTAGACGGAAACGCGGAAGCGGAGAGGGCAGAGGCTCGCTGCTTACATGCGGGGACGTGGAAGAGAACCCCGGTCCGATGGAACGCATTGTGATCTGCCTGATGGTCATCTTCCTGGGCACCTTAGTGCACAAGTCGAGCAGCCAGGGACAGGACAGGCACATGATTAGAATGCGCCAGCTCATCGATATCGTGGACCAGTTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCGGCCCCCGAAGATGTGGAAACCAATTGCGAATGGTCGGCATTTTCCTGCTTTCAAAAGGCACAGCTCAAGTCCGCTAACACCGGGAACAACGAACGGATCATCAACGTGTCCATCAAAAAGCTGAAGCGGAAGCCTCCCTCCACCAACGCCGGACGGAGGCAGAAGCATAGGCTGACTTGCCCGTCATGCGACTCCTACGAGAAGAAGCCGCCGAAGGAGTTCCTGGAGCGGTTCAAGTCGCTCCTGCAAAAGATGATTCATCAGCACCTGTCCTCCCGGACTCATGGGTCTGAGGATTCATGAGGTTAGTCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCTTAGTACTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATTCCGGAATTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAAACCGGTGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCATCTAGAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTAGAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGCTAGCCGGGCTTTTTTTTCTTAGGCCTTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATC CATAGTTGCC

Secretion of IL-12p70 and IL-21 by engineered MSCs are shown in FIG. 45and FIG. 46, respectively, as assessed by ELISA. SB00880 demonstratedexpression of both cytokines by engineered MSCs at higher levels thanthe majority of constructs tested. Additionally, the ratio of IL-12 toIL-21 was determined, as assessed by ELISA and shown in FIG. 47. MSCsengineered using SB00880 demonstrated a 10 fold higher ratio of IL-12p70relative to IL-21. Notably, a ratio of 10:1 has demonstratedpre-clinical efficacy (data not shown).

Functional assays demonstrating expression of IL-12p70 by engineeredMSCs were performed. HEK-293T cells with a STAT4-SEAP reporter, whichreports IL12p70 binding to its receptor and signaling through theJAK-STAT4 pathway, were used to determine potency and activity ofIL12p70 produced by engineered hMSCs. Engineered MSCs were cultured for24 hours and media was collected and incubated with HEK-293T STAT4-SEAPreporter cells. SEAP production was determined with spectrophotometer.As shown in FIG. 48, all constructs that encode IL-12 demonstratedreporter activity indicating functional IL12p70 signaling.

Functional assays demonstrating expression of IL-21 by engineered MSCswere performed. NK-92 human natural killer cells were used to determinefunction of IL-21 produced by engineered hMSCs. Engineered hMSCs werecultured for 24 hours and conditioned media was collected and used totreat NK-92 cells that were deprived from IL-2. Intracellularphospho-flow was performed to quantify phospho-STAT1 and phospho-STAT3activation as a readout for IL-21 activity. As shown in FIG. 49, allconstructs that encode IL-21 demonstrated STAT1 (left panel) and STAT3(right panel) phosphorylation indicating functional IL-21 signaling.

Functional assays for IL-21 was also performed using a IL21R-U2OSIL21R/IL2RG dimerization reporter (PathHunter® U2OS IL21R/IL2RGDimerization Cell Line, DiscoverX Cat. No: 93-1035C3). Reporter cellswere incubated with conditioned media from engineered human MSCs or theappropriate positive (recombinant cytokine) or negative controls. Asshown in FIG. 50, all constructs that encode IL-21 demonstrateddimerization.

Example 20: Engineered MSC Efficacy in CT26 Tumor Model

In the following example, balb/c mMSCs were engineered to express eachof the various murine immune effectors shown in FIG. 51A using thelentiviral transduction method described in Example 6. Each MSC wasengineered to express only a single agent. CT26 tumor cells (5×10⁴ cellsin 100 μl) modified to constitutively express luciferase enzyme (Cat no:CL043, Lot no: CL-IM147 Imanis Life Sciences) were injected into theperitoneal space of immunocompetent balb/c female mice (age 6-8 weeks).One week after tumor implantation, tumor burden was measured byluciferase imaging (BLI) using an AMI imager. Mice were randomized intotreatment groups and treated with intraperitoneally delivered engineeredmMSCs (1×10⁶ cells). MSC-Flag-Myc and PBS were used as a negativecontrol.

As shown in FIG. 51A, significant reductions in tumor burden wereachieved with select effector-producing engineered-MSCs and selecteffector-producing engineered-MSCs in a CT26 syngeneic tumor model.Tumor burden fold change was calculated for each individual mouse bynormalization of post-treatment BLI (day 10) vs pre-treatment BLI. Allthe cases where tumor burden fold change was lower than 1 (dotted line)represent tumor burden reduction. The top MSC-effectors that achievedsignificant reduction in tumor burden were: IL12, IL15, IL12+anti-PD1(microbody), IL12+IL21, IL12+CCL21a, IL12+CXCL10, IL12+CXCL11,IL21+CXCL11, IL21+CCL21a, IL15+CXCL10, GM-CSF+IL12, IL12+IL21+CCL21a.

Example 21: Engineered MSC Efficacy in B16F10 Tumor Model

In the following example, C57BL/6 mMSCs were engineered to express eachof the various murine immune effectors shown in FIG. 51B using thelentiviral transduction method described in Example 6. Each MSC wasengineered to express only a single agent. B16F10 tumor cells (5×10⁴cells in 100 μl) modified to constitutively express luciferase enzyme(B16F10-Fluc-Puro Cat #:CL052, lot #: CL-IM150 Imanis Life Sciences)were injected into the peritoneal space of immunocompetent C57BL/6female mice (age 6-8 weeks). One week after tumor implantation, tumorburden was measured by luciferase imaging (BLI) using an AMI imager.Mice were randomized into treatment groups and treated withintraperitoneally delivered mMSCs 1×10⁶ expressing immune-modulatorycytokines or chemokines such as IL12p70. MSC-Flag-Myc and PBS were usedas a negative control.

As shown in FIG. 51B, significant reductions in tumor burden wereachieved with select effector-producing engineered-MSCs and selecteffector-producing engineered-MSCs in a CT26 syngeneic tumor model.Selected effectors or combinations were achieving significant reductionin tumor burden: IL12, IL12+CD40L, IL12+CXCL10, IL12+IL21,IL12+IL21+Flt3L, IL12+IL21+CXCL10, IL12+CCL21a+Flt3L.

Example 22: IL12 Producing MSCs Reduce CT26 Tumor Burden in an IP Model

In the following example, balb/c mMSCs were engineered to express murineIL12p70 or murine IL21 (i.e., each MSC engineered to express only asingle agent) using the lentiviral transduction method described inExample 6. CT26 tumor cells (5×10⁴ cells in 100 μl) modified toconstitutively express luciferase enzyme (Cat no: CL043, Lot no:CL-IM147 Imanis Life Sciences) were injected into the peritoneal spaceof immunocompetent balb/c (age 6-8 weeks). One week after tumorimplantation, tumor burden was measured by luciferase imaging (BLI)using an AMI imager. In addition, tumor weights were determined at thetime of termination (day 17 post tumor implant). Mice were randomizedinto treatment groups and treated with intraperitoneally delivered mMSCs(1×10⁶ cells). MSC-Flag-Myc and PBS were used as a negative control.Experimental cohorts included: murine IL12-expressing murine MSCs,murine IL21-expressing murine MSCs, and combination treatment of murineIL12-expressing murine MSCs and murine IL21-expressing murine MSCs(1×10⁶ cells delivered for each in the combination).

As shown in FIG. 52A and FIG. 52B, the groups receivingIL12p70-expressing MSCs, IL21-expressing MSCs, and the combination ofIL12p70 and IL21-expressing MSCs led to reduction in tumor burdens asassessed by BLI (FIG. 52A left panel) and by tumor weight (FIG. 52Aright panel) in a CT26 model, including a significant reduction in thecombination treatment, relative to the controls. FIG. 52B demonstratesthe BLI luciferase measurements of individual mice (results summarizedin FIG. 52A left panel).

The above experiment was repeated with the modification of delivering alower dose of engineered mMSCs (1×10⁵ cells). As shown in FIG. 53A, thegroups receiving IL12p70-expressing MSCs, and the combination of IL12p70and IL21-expressing MSCs led to reduction in tumor burdens as assessedby BLI (FIG. 53A; individual BLI measurements of mice—left panel;summary of BLI measurements—right panel) in a CT26 model, including asignificant reduction in the combination treatment, relative to thecontrols. Additionally, the combination treatment demonstrated increasedefficacy relative to groups receiving IL12p70-expressing MSCs alone. Asshown in FIG. 53B, treatment with 1×10⁵ IL12p70 expressing MSCs incombination with 1×10⁵ IL21 expressing MSCs led to tumor-free survivalup to 40 days in all mice treated (n=8; median survival not reached). Incontrast, treatment with 1×10⁵ IL12p70 expressing MSCs alone onlyresulted in a 25% survival rate by day 40 (n=8; median survival 19days). Control groups treated with PBS for FLAG-MSCs resulted in a 0%survival rate by day 40 (n=8 each; median survival 12 days each). Thus,IL21 expression by MSCs enhanced the efficacy of IL12p70 expressingMSCs.

Example 23: IL12 Producing MSCs Reduce B16F10 Tumor Burden in an IPModel

In the following example, C57BL/6 mMSCs were engineered to expressmurine IL12p70 or murine IL21 (i.e., each MSC engineered to express onlya single agent) using the lentiviral transduction method described inExample 6. B16F10 tumor cells (5×10⁴ cells in 100 μl) modified toconstitutively express luciferase enzyme (B16F10-Fluc-Puro Cat #:CL052,lot #: CL-IM150 Imanis Life Sciences) were injected into the peritonealspace of immunocompetent C57BL/6 (age 6-8 weeks). One week after tumorimplantation, tumor burden was measured by luciferase imaging (BLI)using an AMI imager. In addition, tumor weights were determined at thetime of termination (day 17 post tumor implant). Mice were randomizedinto treatment groups and treated with intraperitoneally delivered mMSCs(1×10⁶ cells). MSC-Flag-Myc and PBS were used as a negative control.Experimental cohorts included: murine IL12-expressing murine MSCs,murine IL21-expressing murine MSCs, and combination treatment of murineIL12-expressing murine MSCs and murine IL21-expressing murine MSCs(1×10⁶ cells delivered for each in the combination).

As shown in FIG. 54 and FIG. 55, the groups receiving IL12p70-expressingMSCs, and the combination of IL12p70 and IL21-expressing MSCs led toreduction in tumor burdens as assessed by BLI (FIG. 54 left panel) andby tumor weight (FIG. 54 right panel) in a B16F10 model, including asignificant reduction in the combination treatment, relative to thecontrols. Notably, IL21-expressing MSCs alone did not demonstrate asignificant reduction in tumor burden or tumor weight. FIG. 55demonstrates the BLI luciferase measurements of individual mice for thecontrol FLAG-expressing MSCs and the combination of IL12-expressing MSCsand IL21-expressing MSCs (results summarized in FIG. 54 left panel).

Example 24: MSCs Producing IL12 and IL21 Prolong Tumor-Free Survival ina B16F10 IP Tumor Model and Survive Tumor Rechallenge

In the following example, C57BL/6 mMSCs were engineered to expressmurine IL12 (p70) or murine IL21 (i.e., each MSC engineered to expressonly a single agent) using the lentiviral transduction method describedin Example 6. B16F10 tumor cells (5×10⁴ cells in 100 μl) modified toconstitutively express luciferase enzyme (B16F10-Fluc-Puro Cat #:CL052,lot #: CL-IM150 Imanis Life Sciences) were injected into the peritonealspace of immunocompetent C57BL/6 (age 6-8 weeks). Mice were randomizedinto treatment groups and treated with intraperitoneally delivered mMSCs(1×10⁶ cells). MSC-Flag-Myc and PBS were used as a negative control.Experimental cohorts included: murine IL12-expressing murine MSCs,murine IL21-expressing murine MSCs, and combination treatment of murineIL12-expressing murine MSCs and murine IL21-expressing murine MSCs(1×10⁶ cells delivered for each in the combination).

As shown in FIG. 56, treatment with IL12p70 expressing MSCs led toprolonged survival (median survival 27 days post-treatment) relative tocontrol treated mice (median survival of 8 days post-treatment for bothPBS treated and FLAG-expressing MSCs). Treatment with IL12p70 expressingMSCs in combination with IL21 expressing MSCs led to prolonged survival(54.5% survival; median survival not reached) relative to treatment withIL12p70 expressing MSCs alone. Thus, IL21 expression by MSCs enhancedthe efficacy of IL12p70 expressing MSCs.

Additionally, mice that were tumor-free for more than 90 days weresubsequently re-challenged with B16-F10 tumor cells implanted in theflank (1×10⁶ cells). Naïve un-treated mice were implanted at the sametime as controls. Sub-cutaneous tumor burden was measured by caliper. Asshown in FIG. 57C, all mice (n=4) that previously received thecombination treatment of IL12-expressing MSCs and IL21-expressing MSCssurvived rejected the newly implanted tumor, indicating that thetreatment resulted in achievement of anti-tumor immune memory. Mice thatpreviously received the treatment of IL12-expressing MSCs alone had a50% tumor-rejection rate (2 out of 4 mice; FIG. 57B). In contrast, tumorwere established in 60% of naïve mice (3 out of 5 mice; FIG. 57A).

Example 25: MSCs Producing IL12 in Combination with Immune CheckpointTherapy Prolong Survival in a B16F10 IP Tumor Model

In the following example, C57BL/6 mMSCs were engineered to expressmurine IL12 (p70) using the lentiviral transduction method described inExample 6. B16F10 tumor cells (5×10⁴ cells in 100 μl) modified toconstitutively express luciferase enzyme (B16F10-Fluc-Puro Cat #:CL052,lot #: CL-IM150 Imanis Life Sciences) were injected into the peritonealspace of immunocompetent C57BL/6 (age 6-8 weeks). Mice were randomizedinto treatment groups and treated with IP administration of anti-PD1antibody (clone RMP1-14) at a dose of 200 mg/kg alone or in combinationwith low dose (1e5) of IL12-expressing murine MSCs.

As shown in FIG. 56, treatment with anti-PD1 alone resulted in a 12.5%survival rate and median survival of 23 days (FIG. 56 “Anti-PD1”; 1 outof 8 mice had long term tumor-free survival). In contrast, the combinedtreatment of anti-PD1 with IL12p70 expressing MSCs resulted in a 50%survival rate (FIG. 56 “MSC-IL12 (p70)+Anti-PD1”; 4 out of 8 mice hadlong term tumor-free survival; median survival not yet established).Thus, IL12 expression by MSCs enhanced the efficacy of anti-PD1 immunecheckpoint therapy and convert a checkpoint refractory or resistantmodel (B16F10) into responsive.

Example 26: MSCs Producing Both IL12 and IL21 Reduce Tumor Burden in aCT26 IP Tumor Model

In the following example, balb/c mMSCs were engineered to express murineIL12 (p70) and murine IL21 from a single lentiviral expression vector.The lentiviral expression vector used a 2A ribosome skipping elements toexpress both cytokines from a single transcript using the lentiviraltransduction method described in Example 6. CT26 tumor cells (1×10⁵cells in 100 μl) modified to constitutively express luciferase enzyme(Cat no: CL043, Lot no: CL-IM147 Imanis Life Sciences) were injectedinto the peritoneal space of immunocompetent female balb/c mice (age 6-8weeks). One week after tumor implantation, tumor burden was measured byluciferase imaging (BLI) using an AMI imager. Mice were randomized intotreatment groups and treated IP with different amounts of mMSCs rangingfrom 1×10⁴ to 1×10⁶ cells. MSC-Flag-Myc (1×10⁶ cells) and PBS were usedas a negative control.

As shown in FIG. 58A-C, anti-tumor activity was observed in adose-dependent manner of MSCs expressing both IL12 and IL21, as assessedby BLI (FIG. 58A normalized day 17 vs day 7; FIG. 58B and FIG. 58C BLIover time for individual mice). No efficacy was observed in control FLAGor PBS mice (FIG. 58A and FIG. 58B). In contrast, minimal efficacy wasobserved at a dose of 1×10⁴, with efficacy increasing at each increaseddose (FIG. 58A and FIG. 58C). As shown in FIG. 58D, long term tumor-freesurvival up to 60 days post tumor implant was observed in adose-dependent manner, with mice treated with 1×10⁶ to 1×10⁵ havingsignificantly extended tumor-free survival (Median survivalpost-implant: PBS/FLAG—19 days; 1×10⁶ to 1×10⁵—not reached; 3×10⁴—53days; 1×10⁴—18-19 days).

Example 27: MSCs Producing Both IL12 and IL21 Reduce Tumor Burden in aB16F10 IP Tumor Model

In the following example, C57BL/6 mMSCs were engineered to expressmurine IL12 (p70) and murine IL21 from a single lentiviral expressionvector. The lentiviral expression vector used a 2A ribosome skippingelements to express both cytokines from a single transcript using thelentiviral transduction method described in Example 6. B16F10 tumorcells (5×10⁴ cells in 100 μl) modified to constitutively expressluciferase enzyme (B16F10-Fluc-Puro Cat #:CL052, lot #: CL-IM150 ImanisLife Sciences) were injected into the peritoneal space ofimmunocompetent C57BL/6 (age 6-8 weeks). One week after tumorimplantation, tumor burden was measured by luciferase imaging (BLI)using an AMI imager. Mice were randomized into treatment groups andtreated with different amounts of mMSCs ranging from 1×10⁵ to 1×10⁷cells). MSC-Flag-Myc (3×10⁶ cells) and PBS were used as a negativecontrol. Some groups were treated with multiple doses separated by 5days (treatment on day 7, 12 and 17 post tumor-implant).

As shown in FIG. 59A-D, anti-tumor activity was observed in adose-dependent manner of MSCs expressing both IL12 and IL21, as assessedby BLI (FIG. 59A normalized day 17 vs day 7; FIG. 59B-D BLI over timefor individual mice). No efficacy was observed in control FLAG or PBSmice (FIG. 59A and FIG. 59B). No efficacy was also observed at doses of1×10⁵ or 3×10⁵ cells (FIG. 59A and FIG. 59C). In contrast, minimalefficacy was observed at a dose of 1×10⁶, with efficacy increasing ateach increased dose (FIG. 59A and FIG. 59C). Efficacy was also observedfollowing multiple administrations of higher doses (FIG. 59D). As shownin FIG. 59E, long term tumor-free survival was observed in adose-dependent manner, and also observed following multipleadministrations of higher doses (Median survival post-implant: PBS—20days; FLAG (×3)—27 days; 1×10⁷ —31.5 days; 3×10⁶ —36 days; 3×10⁶ (×3)—39days; 1×10⁶ —33 days; 1×10⁶ (×3)—39 days; 3×10⁵ —27 days; 3×10⁵ (×3)—27days [curve overlaps with 3×10⁵ treatment]; 1×10⁵—24 days).

Example 28: MSCs Producing Both IL12 and IL21 Reduce Tumor Burden in aMC-38 IP Tumor Model

In the following example, C57BL/6 mMSCs were engineered to expressmurine IL12 (p70) and murine IL21 from a single lentiviral expressionvector. The lentiviral expression vector used a 2A ribosome skippingelements to express both cytokines from a single transcript using thelentiviral transduction method described in Example 6. MC-38 tumor cellswere transduced with fLUC-EGFP construct and sorted based on EGFPfluorescence, then 5×10⁵ cells were injected into the peritoneal spaceof immunocompetent C57BL/6 (age 6-8 weeks). Nine days after tumorimplantation, tumor burden was measured by luciferase imaging (BLI)using an AMI imager. Mice were randomized into treatment groups andtreated with different amounts of mMSCs ranging from 3×10⁴ to 1×10⁶cells. MSC-Flag-Myc and PBS were used as a negative control.

As shown in FIG. 60A and FIG. 60B, anti-tumor activity was observed in adose-dependent manner of MSCs expressing both IL12 and IL21, as assessedby BLI (FIG. 60A normalized day 18 vs day 9; FIG. 60B BLI over time forindividual mice). No efficacy was observed in control FLAG or PBS mice(FIG. 60A and FIG. 60B). No efficacy was also observed at doses of 1×10⁵or 3×10⁴ cells (FIG. 60A and FIG. 60B). In contrast, minimal efficacywas observed at a dose of 3×10⁵, with efficacy increasing at anincreased dose of 1×10⁶ cells (FIG. 60A and FIG. 60B). As shown in FIG.60C, long term tumor-free survival was observed in a dose-dependentmanner, with all mice treated with 1×10⁶ cells surviving past at leastday 30 (Median survival post-implant: PBS—21 days; FLAG—29 days;1×10⁶—not reached; 3×10⁵—28 days; 1×10⁵—21 days; 3×10⁴—21 days [PBS,1×10⁵, and 3×10⁴ overlap). Accordingly, mMSCs engineered to expressmurine IL12 (p70) and murine IL21 demonstrated efficacy in a MC-38 tumormodel.

Example 29: Human MSCs Home to Tumors in an IP Model

In the following example, NSG mice were implanted with OVCAR8-fLUC cellsIP. 14-21 days after tumor implantation, 1×10⁶ human BM-MSCs engineeredto express Nanoluc-EGFP were delivered IP. Mice were euthanized at 24hours post injection of MSCs and peritoneal organs (stomach, kidney,liver, colon, spleen, pancreas, omentum/tumor, ovaries and Fallopiantubes) were imaged ex-vivo for NanoLuc signaling (NanoGlo Substrate Kit,Vendor: Promega, Catalog No.: N1110). Human MSCs were imaged by EGFPfluorescence in tumor sections collected at 24 hours as well as 22 dayspost injection.

As shown in FIG. 61A and FIG. 61B, human MSC NanoLuc signal waspreferentially enriched in the tumor compared to the other organs in theperitoneal cavity (FIG. 61A summarized luciferase quantification; FIG.61B representative images of luciferase signal). Additionally,persistence of MSCs was lower than 22 days, with no cells being detectedat the latest time point (FIG. 61B right most panel).

Example 30: Biodistribution and PK of Effector Cytokines

In the following example, biodistribution and PK of effector cytokinesproduced by engineered MSCs was assessed.

In a first experiment, NSG mice were implanted with 5×10⁶ OVCAR8-fLUCtumor cells IP. 21-27 days after tumor implantation, mice wererandomized based on tumor burden measured by BLI and treated with 1×10⁶hMSCs engineered to express human IL12 (p70) and human IL21 from asingle lentiviral expression vector. The lentiviral expression vectorused a 2A ribosome skipping elements to express both cytokines from asingle transcript using the lentiviral transduction method described inExample 6. Mice were euthanized at 16-24 hours or 3, 4 and 7 days postMSC treatment and peritoneal fluid was collected via IP lavage byinjecting 1 mL of PBS into the peritoneal space and collecting it. Serumwas separated from whole blood after intracardiac puncture. ELISA (R&Dsystems) was used to determine the protein amount in each compartment(peritoneal fluid vs serum) for each time point and treatment type.

As shown in FIG. 62A, transient production of both human IL12 (leftpanel) and human IL21 (right panel) was observed in both the peritonealfluid (left column for each respective time point) and serum (rightcolumn for each respective time point). At least a 10 fold increasedprotein abundance was observed in the peritoneal space (local) comparedto systemic (serum), demonstrating localized delivery of cytokines byengineered MSCs.

In another experiment, balb/c mMSCs were engineered to express murineIL12p70 or murine IL21 (i.e., each MSC engineered to express only asingle agent) using the lentiviral transduction method described inExample 6. CT26-fLUC tumor cells (1×10⁵ cells in 100 μl) were injectedinto the peritoneal space of immunocompetent balb/c (age 6-8 weeks).Murine IL12-expressing murine MSCs and murine IL21-expressing murineMSCs (1×10⁶ cells delivered for each in the combination) were deliveredIP. Mice were euthanized at 24 or 72 hours post MSC treatment andperitoneal fluid was collected via IP lavage by injecting 1 mL of PBSinto the peritoneal space and collecting it. Serum was separated fromwhole blood after intracardiac puncture. Luminex (Millipore) was used todetermine the protein amount in each compartment (peritoneal fluid vsserum) for each time point and treatment type.

As shown in FIG. 62B, transient production of both murine IL12 (leftpanel) and murine IL21 (right panel) was observed in both the peritonealfluid (left column for each respective time point) and serum (rightcolumn for each respective time point). At least a 10 fold increasedprotein abundance was observed in the peritoneal space (local) comparedto systemic (serum), demonstrating localized delivery of cytokines byengineered MSCs.

Example 31: Comparison of MSC Treatment and Recombinant CytokineTreatment in a CT26 IP Tumor Model

In the following example, balb/c mMSCs were engineered to express murineIL12 (p70) and murine IL21 from a single lentiviral expression vector.Balb/c mMSCs were also engineered to express either murine IL12 (p70) ormurine IL21. The lentiviral expression vector used a 2A ribosomeskipping elements to express both cytokines from a single transcriptusing the lentiviral transduction method described in Example 6. CT26tumor cells (1×10⁵ cells in 100 μl) modified to constitutively expressluciferase enzyme (Cat no: CL043, Lot no: CL-IM147 Imanis Life Sciences)were injected into the peritoneal space of immunocompetent female balb/cmice (age 6-8 weeks). One week after tumor implantation, tumor burdenwas measured by luciferase imaging (BLI) using an AMI imager. For MSCtreated mice, mice were randomized into treatment groups and treatedwith intraperitoneally delivered mMSCs (1×10⁶ cells), receiving murineIL12-expressing murine MSCs, murine IL21-expressing murine MSCs, ormurine IL12 and IL21-expressing murine MSCs, with MSC-Flag-Myc and PBSwere used as a negative control. Additionally, treatment groups alsoincluded mice receiving a bolus dose of the respective recombinantcytokines at a dose of 4-times the amount produced by MSCs in vitro(measured by ELISA—recombinant IL12: 5 ug/mouse; Recombinant IL21: 0.4ug/mouse). Tumor burden was measured by fLUC BLI across time points andmice were euthanized when reaching endpoint criteria due to tumorburden. Kaplan Meier survival curves were determined to calculatetumor-free survival.

As shown in FIG. 63A-C, mice treated with MSCs engineered to producecytokines outperformed recombinant cytokine therapy in terms ofprolonged tumor-free survival, in all cases (FIG. 63A—MSC-IL12 vs rIL12;FIG. 63B—MSC-IL21 vs rIL21; FIG. 63C—MSC-IL12/IL21 vs rIL12+rIL21).Additionally, as shown in FIG. 63D-E, mice treated with MSCs engineeredto produce cytokines outperformed recombinant cytokine therapy asassessed by tumor burden BLI), in all cases (FIG. 63D bottomrow—MSC-IL12 vs rIL12; FIG. 63E top row—MSC-IL21 vs rIL21; FIG. 63Ebottom row—MSC-IL12/IL21 vs rIL12+rIL21).

Example 32: Comparison of MSC Treatment and Recombinant CytokineTreatment in a B16F10 IP Tumor Model

In the following example, C57BL/6 mMSCs were engineered to expressmurine IL12 (p70) and murine IL21 from a single lentiviral expressionvector. C57BL/6 mMSCs were also engineered to express either murine IL12(p70) or murine IL21. The lentiviral expression vector used a 2Aribosome skipping elements to express both cytokines from a singletranscript using the lentiviral transduction method described in Example6. B16F10 tumor cells (1×10⁵ cells in 100 μl) modified to constitutivelyexpress luciferase enzyme (Cat no: CL043, Lot no: CL-IM147 Imanis LifeSciences) were injected into the peritoneal space of immunocompetentfemale balb/c mice (age 6-8 weeks). One week after tumor implantation,tumor burden was measured by luciferase imaging (BLI) using an AMIimager. For MSC treated mice, mice were randomized into treatment groupsand treated with intraperitoneally delivered mMSCs (3×10⁶ cells)engineered to express both IL12 and IL21-expressing murine MSCs, withMSC-Flag-Myc and PBS were used as a negative control. Additionally,treatment groups also included mice receiving a bolus dose of therespective recombinant cytokines at a dose of 4-times the amountproduced by MSCs in vitro (measured by ELISA—recombinant IL12: 3ug/mouse; Recombinant IL21: 0.03 ug/mouse). Tumor burden was measured bytumor weight at day 7 post treatment and mice were euthanized whenreaching endpoint criteria due to tumor burden. Kaplan Meier survivalcurves were determined to calculate tumor-free survival.

As shown in FIG. 64A, mice treated with MSCs engineered to produce bothIL12 and IL21 outperformed recombinant cytokine therapy as assessed bytumor weight. Additionally, as shown in FIG. 64B, mice treated with MSCsengineered to produce both IL12 and IL21 outperformed recombinantcytokine therapy as assessed by tumor-free prolonged survival.

Example 33: Immune Profile Following Treatment with MSCs Producing BothIL12 and IL21 in a CT26 IP Tumor Model

In the following example, balb/c mMSCs were engineered to express murineIL12p70 or murine IL21 (i.e., each MSC engineered to express only asingle agent) using the lentiviral transduction method described inExample 6. CT26 tumor cells (1×10⁵ cells) modified to constitutivelyexpress luciferase enzyme (Cat no: CL043, Lot no: CL-IM147 Imanis LifeSciences) were injected into the peritoneal space of immunocompetentbalb/c (age 6-8 weeks). One week after tumor implantation, tumor burdenwas measured by luciferase imaging (BLI) using an AMI imager. Mice wererandomized into treatment groups and treated with a intraperitoneallydelivered combination treatment of murine IL12-expressing murine MSCsand murine IL21-expressing murine MSCs (1×10⁶ cells delivered for eachin the combination), or MSC-Flag-Myc and PBS as a negative controls.Mice were euthanized and organs collected at 72 hours after treatment.Multicolor flow cytometry was used to characterize immune infiltrates inresponse to treatment.

As shown in FIG. 65A and FIG. 65B, T-cell subsets and activation markers(CD3, CD4, CD8, CD8/CD38+, CD8/IFNg+, CD8/Gzmb+, NK/Gzmb+ and ratioCD8:Tregs-FoxP3) were significantly increased in the peritoneal fluidafter treatment with MSCs-IL12+MSCs IL21. Additionally, as shown in FIG.65C, antigen-presenting cells such as dendritic cells (CD11c/MHC-II hi,CD86+, CD103+, CD11b+) were also significantly increased in peritonealtumor-draining lymph nodes after treatment with MSC-IL12+MSC-IL21.Accordingly, combination treatment of murine IL12-expressing murine MSCsand murine IL21-expressing murine MSCs demonstrated an activated immuneprofile.

Example 34: Optimization of Signal Peptide Sequences

In the following example, effector molecules are modified to replacetheir native signal peptide sequence with an exogenous signal peptidesequence (see Table 5 for exemplary signal peptide sequences that aretested). Modified effector molecules are tested for functionalimprovements such as improved expression and maintained secretion, suchas in particular environments (e.g., tumor microenvironments).Functional performance for the modified effector molecules is alsotested in tumor models (e.g., improved ability to clear tumors, improvedability to clear tumors in different environments, or improved abilityto clear different types of tumors).

Example 35: Enrichment of Engineered MSCs

In the following example, MSCs are engineered to express effectormolecules within a population of cells that include unmodified cells,such as unmodified MSCs. The engineered MSCs are enriched within thepopulation by contacting the engineered MSCs with a growth factor (suchas the effector molecules described in Table 1) such that thoseengineered MSCs that are enriched are a sub-population of engineeredMSCs that express a receptor or receptor ligand for the growth factor.The sub-population of engineered MSCs of interest are contacted with thegrowth factor in various manners:

-   -   1. In an autocrine manner by genetically engineering the MSCs        themselves to express the factors.    -   2. In a paracrine manner by genetically engineering feeder or        support cells to express the factors and supply those factors to        the MSCs, or by using conditioned media containing the factors        from the feeder or support cells (such as 293 Ts) engineered to        express these factors.    -   3. In an endocrine manner, by injecting recombinant protein or        nucleic acid versions of these factors into patients following        MSC transplantation.    -   4. Via addition of soluble recombinant protein versions of these        factors to the MSC culture conditions.    -   5. Via coating of the tissue culture plate/flask surfaces used        for MSC propagation with recombinant versions of these factors.

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All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A method of treating a human subject havingcancer, wherein the method comprises delivering to the subject anengineered human cell in an effective amount to induce an immuneresponse, wherein the engineered human cell comprises: a) a promoter;and b) an exogenous polynucleotide sequence comprising an expressioncassette described in a formula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2 wherein S1 comprises a polynucleotide sequence encoding afirst signal peptide, E1 comprises a polynucleotide sequence encoding afirst effector molecule, L comprises a linker polynucleotide sequence,S2 comprises a polynucleotide sequence encoding a second signal peptide,E2 comprises a polynucleotide sequence encoding a second effectormolecule, and wherein the promoter is operably linked to the expressioncassette, the first signal peptide is operably linked to the firsteffector molecule, and the second signal peptide is operably linked tothe second effector molecule, wherein the engineered human cell isselected from the group consisting of: a mesenchymal stem cell (MSC),natural killer (NK) cell, NKT cell, and macrophage, wherein (a) thefirst effector molecule comprises an IL12p70 fusion protein and thesecond effector molecule comprises CCL21a, IL7, IL15, IL21, Flt3L, ananti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein, (b) thefirst effector molecule comprises CCL21a, IL7, IL15, IL21, Flt3L, ananti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein and thesecond effector molecule comprises an IL12p70 fusion protein, (c) thefirst effector molecule comprises IL21 and the second effector moleculecomprises CCL21a, IL7, IL15, IL18, an IL12p70 fusion protein, Flt3L, ananti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein, or (d) thefirst effector molecule comprises CCL21a, IL7, IL15, IL18, an IL12p70fusion protein, Flt3L, an anti-PD1 antibody, CD40L, or a CXCL10-CXCL11fusion protein and the second effector molecule comprises IL21, whereinat least one of the first effector molecule and the second effectormolecule does not comprise, nor is operably linked to, a transmembranedomain, and wherein the engineered human cell further comprises achimeric antigen receptor (CAR) or exogenous polynucleotide sequenceencoding the same.
 2. The method of claim 1, wherein the promoter isoperably linked to the expression cassette such that the polynucleotidesare capable of being transcribed as a single polynucleotide comprisingthe formula S1-E1-L-S2-E2.
 3. The method of claim 1, wherein the linkerpolynucleotide sequence is operably associated with the translation ofthe first effector molecule and the second effector molecule as separatepolypeptides.
 4. The method of claim 3, wherein the linkerpolynucleotide sequence encodes a 2A ribosome skipping tag or encodes anInternal Ribosome Entry Site (IRES), optionally wherein when the linkerpolynucleotide sequence encodes a 2A ribosome skipping tag, and the 2Aribosome skipping tag is selected from the group consisting of: P2A,T2A, E2A, and F2A.
 5. The method of claim 1, wherein the linkerpolynucleotide sequence encodes a second promoter, wherein the promoteris operably linked to the expression cassette such that a firstpolynucleotide comprising the formula S1-E1 is capable of beingtranscribed, wherein the second promoter is operably linked to theexpression cassette such that a second polynucleotide comprising theformula S2-E2 is capable of being transcribed, and wherein the first andthe second polynucleotide are separate polynucleotides.
 6. The method ofclaim 1, wherein the engineered human cell is a natural killer (NK)cell.
 7. The method of claim 1, wherein the promoter comprises aconstitutive promoter selected from the group consisting of: CMV, EFS,SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1,hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.
 8. The method of claim1, wherein the promoter comprises an inducible promoter selected fromthe group consisting of: minP, NFkB response element, CREB responseelement, NFAT response element, SRF response element 1, SRF responseelement 2, AP1 response element, TCF-LEF response element promoterfusion, Hypoxia responsive element, SMAD binding element, STAT3 bindingsite, inducer molecule responsive promoters, and tandem repeats thereof.9. The method of claim 1, wherein: the first signal peptide comprises anative signal peptide native to the first effector molecule; or thesecond signal peptide comprises a native signal peptide native to thesecond effector molecule; or the first signal peptide comprises anon-native signal peptide non-native to the first effector molecule; orthe second signal peptide comprises a non-native signal peptidenon-native to the second effector molecule, respectively; or anycombination thereof.
 10. The method of claim 1, wherein the expressioncassette further comprises following E2, an additional exogenouspolynucleotide sequence comprising a formula, oriented from 5′ to 3′,comprising: (L-S-E)_(x) wherein S comprises a polynucleotide sequenceencoding a signal peptide, E comprises a polynucleotide sequenceencoding an additional effector molecule, L comprises a linkerpolynucleotide sequence, X=1 to 20 wherein the promoter is operablylinked to the expression cassette, and wherein for each iteration for agiven X, the corresponding signal peptide is operably linked to therespective effector molecule, optionally wherein one or more of theadditional effector molecules comprises a chimeric antigen receptor. 11.The method of claim 1, wherein the exogenous polynucleotide sequencecomprises one or more viral vector polynucleotide sequences, and whereinthe one or more viral vector polynucleotide sequences compriselentiviral, retroviral, retrotransposon, or adenoviral polynucleotidesequences.
 12. A method of reducing tumor volume in a human subject, themethod comprising delivering to the subject having a tumor a compositioncomprising an engineered human cell comprising: a) a promoter; and b) anexogenous polynucleotide sequence comprising an expression cassettedescribed in a formula, oriented from 5′ to 3′, comprisingS1-E1-L-S2-E2 wherein S1 comprises a polynucleotide sequence encoding afirst signal peptide, E1 comprises a polynucleotide sequence encoding afirst effector molecule, L comprises a linker polynucleotide sequence,S2 comprises a polynucleotide sequence encoding a second signal peptide,E2 comprises a polynucleotide sequence encoding a second effectormolecule, and wherein the promoter is operably linked to the expressioncassette, the first signal peptide is operably linked to the firsteffector molecule, and the second signal peptide is operably linked tothe second effector molecule, and wherein the engineered human cell isselected from the group consisting of: a mesenchymal stem cell (MSC),natural killer (NK) cell, NKT cell, and macrophage, wherein (a) thefirst effector molecule comprises an IL12p70 fusion protein and thesecond effector molecule comprises CCL21a, IL7, IL15, IL21, Flt3L, ananti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein, (b) thefirst effector molecule comprises CCL21a, IL7, IL15, IL21, Flt3L, ananti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein and thesecond effector molecule comprises an IL12p70 fusion protein, (c) thefirst effector molecule comprises IL21 and the second effector moleculecomprises CCL21a, IL7, IL15, IL18, an IL12p70 fusion protein, Flt3L, ananti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein, or (d) thefirst effector molecule comprises CCL21a, IL7, IL15, IL18, an IL12p70fusion protein, Flt3L, an anti-PD1 antibody, CD40L, or a CXCL10-CXCL11fusion protein and the second effector molecule comprises IL21, andwherein at least one of the first effector molecule and the secondeffector molecule does not comprise, nor is operably linked to, atransmembrane domain, and wherein the engineered human cell furthercomprises a chimeric antigen receptor (CAR) or exogenous polynucleotidesequence encoding the same.
 13. A method of inducing an immune responsein a human subject, the method comprising administering atherapeutically effective dose of an engineered human cell comprising:a) a promoter; and b) an exogenous polynucleotide sequence comprising anexpression cassette described in a formula, oriented from 5′ to 3′,comprisingS1-E1-L-S2-E2 wherein S1 comprises a polynucleotide sequence encoding afirst signal peptide, E1 comprises a polynucleotide sequence encoding afirst effector molecule, L comprises a linker polynucleotide sequence,S2 comprises a polynucleotide sequence encoding a second signal peptide,E2 comprises a polynucleotide sequence encoding a second effectormolecule, and wherein the promoter is operably linked to the expressioncassette, the first signal peptide is operably linked to the firsteffector molecule, and the second signal peptide is operably linked tothe second effector molecule, and wherein the engineered human cell isselected from the group consisting of: a mesenchymal stem cell (MSC),natural killer (NK) cell, NKT cell, and macrophage, wherein (a) thefirst effector molecule comprises an IL12p70 fusion protein and thesecond effector molecule comprises CCL21a, IL7, IL15, IL21, Flt3L, ananti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein, (b) thefirst effector molecule comprises CCL21a, IL7, IL15, IL21, Flt3L, ananti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein and thesecond effector molecule comprises an IL12p70 fusion protein, (c) thefirst effector molecule comprises IL21 and the second effector moleculecomprises CCL21a, IL7, IL15, IL18, an IL12p70 fusion protein, Flt3L, ananti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein, or (d) thefirst effector molecule comprises CCL21a, IL7, IL15, IL18, an IL12p70fusion protein, Flt3L, an anti-PD1 antibody, CD40L, or a CXCL10-CXCL11fusion protein and the second effector molecule comprises IL21, andwherein at least one of the first effector molecule and the secondeffector molecule does not comprise, nor is operably linked to, atransmembrane domain, and wherein the engineered human cell furthercomprises a chimeric antigen receptor (CAR) or exogenous polynucleotidesequence encoding the same.
 14. The method of claim 1, wherein the firsteffector molecule comprises an IL12p70 fusion protein and the secondeffector molecule comprises CCL21a, IL7, IL15, IL21, Flt3L, an anti-PD1antibody, CD40L, or a CXCL10-CXCL11 fusion protein.
 15. The method ofclaim 1, wherein the first effector molecule comprises CCL21a, IL7,IL15, IL21, Flt3L, an anti-PD1 antibody, CD40L, or the CXCL10-CXCL11fusion protein and the second effector molecule comprises the IL12p70fusion protein.
 16. The method of claim 1, wherein the first effectormolecule comprises IL21 and the second effector molecule comprises IL15.17. The method of claim 13, wherein the first effector moleculecomprises the IL12p70 fusion protein and the second effector moleculecomprises CCL21a, IL7, IL15, IL21, Flt3L, an anti-PD1 antibody, CD40L,or a CXCL10-CXCL11 fusion protein.
 18. The method of claim 13, whereinthe first effector molecule comprises CCL21a, IL7, IL15, IL21, Flt3L, ananti-PD1 antibody, CD40L, or a CXCL10-CXCL11 fusion protein and thesecond effector molecule comprises the IL12p70 fusion protein.
 19. Themethod of claim 13, wherein the first effector molecule comprises IL21and the second effector molecule comprises IL15.
 20. The method of claim13, wherein the engineered human cell is a natural killer (NK) cell.